Vascular endothelial growth factor antagonists and methods for their use

ABSTRACT

The present invention provides variant VEGF polypeptides which have been altered in their C-terminal heparin binding region to lower their heparin binding affinity. These variants have been found to act as receptor antagonists for VEGF receptors and antagonize angiogenesis. These variants are useful to treat diseases characterized by pathological angiogenesis.

STATEMENT REGARDING GOVERNMENT RIGHTS

The instant application and the invention(s) described herein are theproperty of the United States Government. The work related to thispatent application was funded, at least in part by, the NationalInstitutes of Health, as part of project 1ZIABC011124.

SEQUENCE LISTING

The Sequence Listing text file attached hereto, created Apr. 22, 2013,size 156 kilobytes, and filed herewith as file name“6137NCI31PCT_SEQ_(—)20120427_ST25.txt” is incorporated herein byreference in its entirety.

BACKGROUND

Vascular endothelial growth factor-A (VEGF-A) is an essential regulatorof angiogenesis during embryogenesis and adulthood, and mediatespathological angiogenesis in many diseases, including cancer. VEGF-Asignals through the receptor tyrosine kinases VEGFR1 and VEGFR2; theVEGFA gene encodes multiple VEGF-A isoforms, all of which bind VEGFR1and VEGFR2, but differ in their capacity to bind to heparan sulfateproteoglycans (HSP) also present on target cells. HSP binding plays acritical role of VEGF-A signaling; mice engineered to express only anon-HSP binding isoform display defective microvessel branching andfrequently die shortly after birth.

Vascular endothelial growth factor is a member of the PDGF family thatis characterized by the presence of eight conserved cysteine residuesand a cystine knot structure. Humans express alternately splicedisoforms of 121, 145, 165, 183, 189, and 206 amino acids in length.VEGF165 appears to be the most abundant and potent isoform, followed byVEGF121 and VEGF189. Isoforms other than VEGF121 contain basic heparinbinding regions and are not freely diffusible.

Human VEGF165 shares 88% amino acid sequence identity with correspondingregions of mouse and rat, 96% with porcine, 95% with canine, and 93%with feline, equine and bovine VEGF, respectively. VEGF binds the type Itransmembrane receptor tyrosine kinases VEGF R1 (also called Flt1) andVEGF R2 (Flk1/KDR) on endothelial cells. Although VEGF affinity ishighest for binding to VEGF R1, VEGF R2 appears to be the primarymediator of VEGF angiogenic activity.

Within the carboxyl terminal domain of the VEGF165 isoform, amino acidsR123, R124, and R159 are reportedly critical for HSP binding. However,it is noted that replacement of these amino acids with alanine causesreduced binding of VEGF 165 to VEGFR1 but does not affect binding ofVEGF 165 to VEGFR2. Importantly, in an in vitro assay which assessesangiogenic activity, alanine substitution mutants of native VEGF showedthe same ability to enhance angiogenesis as native VEGF. Alaninesubstituted mutants also showed reduced affinity for heparin, heparansulfate proteoglycans, and for the VEGFR2 coreceptor, NRP1. VEGFR2 hasbeen identified as the key signaling receptor that mediates theproliferative and migratory effects of VEGF. Alanine mutants were foundto possess an angiogenic activity as potent as that of native VEGF154(mouse), indicating that VEGF carboxyl-terminal domain is not directlyinvolved in VEGFR2 binding.

The critical role of VEGF signaling in many human cancers, particularlyas a driver of tumor growth and metastasis, for example, has made VEGFand its receptors important anti-cancer drug targets. VEGF signalingblockade has been achieved using VEGFR ATP binding site antagonists andVEGF neutralizing antibodies; each of these approaches has features aswell as limitations. Neutralizing antibodies are highly selective fortheir intended target, e.g. VEGF, and have few, if any, off-targeteffects. ATP binding antagonists, in contrast, are often cheaper tomanufacture than antibodies, but are less selective for their intendedtarget and off-target toxicities are frequently associated with theiruse. Biological antagonists derived from the native protein ligand, e.g.VEGF, can be more cost-effective to produce than larger antibodymolecules, yet possess a comparable level of target selectivity. Areceptor antagonist would be desirable because a receptor antagonistwould bind to the receptor, and would be likely to exert inhibitoraction even where receptor activation is independent of VEGF binding.Bevacizumab (Avastin) is the major biological VEGF antagonistanti-cancer therapeutic approved so far by the US FDA. Bevacizumab isexpensive to produce because it requires mammalian expression formanufacture, the most expensive recombinant expression system used inprotein production. Aflibercept, a VEGFR fusion protein has US FDAapproval for the treatment of colorectal cancer and wet maculardegeneration. Among existing VEGF antagonists, Aflibercept has a uniquetarget spectrum, although it too is manufactured using a mammalian cellexpression system. It would be desirable to manufacture such anantagonist in a less expensive system, for example, via P. pastoris.

SUMMARY

In one embodiment, the present invention includes a method for treatinga disease characterized by pathological angiogenesis. This methodincludes administering to a patient in need thereof a pharmaceuticallyeffective amount of a vascular endothelial cell growth factor (VEGF)polypeptide comprising a variant C-terminal heparin binding domain and anative receptor tyrosine kinase binding domain. In one embodiment thepolypeptide comprises a variant C-terminal heparin domain. This variantpolypeptide has one or more amino acid alternations from a native VEGFpolypeptide designed to occupy the receptor and repel and/or reducebinding affinity to heparin, rather than bind heparin and/or heparansulfate containing proteoglycans with high affinity, as does nativeVEGF. In one embodiment, the affinity of the variant polypeptide forboth VEGFR-1 (FLT-1) and VEGFR-2 (KDR/FLK-1) is substantially maintainedin comparison to said native VEGF.

In another embodiment, the variant polypeptide antagonizes KDR signalactivation. In one embodiment, the native VEGF polypeptide can be one ofthe following: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

The present invention includes variant VEGF polypeptides. A vascularendothelial cell growth factor (VEGF) variant polypeptide includespolypeptides comprising a variant C-terminal heparin binding domain, anda native receptor tyrosine kinase binding domain, wherein said variantpolypeptide has one or more amino acid alternations from a native VEGFpolypeptide, wherein said variant polypeptide binds heparin at a loweraffinity than and/or repels heparin as compared to said native VEGF, andwherein the affinity of the variant polypeptide for both VEGFR-1 (FLT-1)and VEGFR-2 (KDR/FLK-1) is substantially maintained in comparison tosaid native VEGF, and wherein said variant polypeptide antagonizes KDRsignal activation. In one embodiment the polypeptide comprises a variantC-terminal heparin domain.

A variant polypeptide of the invention can be any of the following: apolypeptide which includes SEQ ID NO:25 wherein the amino acid of atleast one of positions 149, 150, and 185 of SEQ ID NO:25 is an acidicamino acid, a polypeptide comprising SEQ ID NO:26 wherein the amino acidof at least one of positions 123, 124, and 159 of SEQ ID NO:26 is anacidic amino acid, or a polypeptide comprising SEQ ID NO:27 wherein theamino acid of at least one of positions 13, 14, and 49 of SEQ ID NO:27is an acidic amino acid; or a polypeptide having at least 95% identityto any of the above polypeptides and having the ability to antagonizeKDR signal activation. In one embodiment, the acidic amino acid is E. Inanother embodiment, the variant polypeptide can be any of the following:a polypeptide comprising SEQ ID NO:25 wherein the amino acid atpositions 149, 150, and 185 of SEQ ID NO:25 is an acidic amino acid, apolypeptide comprising SEQ ID NO:26 wherein the amino acid at positions123, 124, and 159 of SEQ ID NO:26 is an acidic amino acid, or apolypeptide comprising SEQ ID NO:27 wherein the amino acid at positions13, 14, and 49 of SEQ ID NO:27 is an acidic amino acid; or a polypeptidehaving at least 95% identity to any of the preceding polypeptides andhaving the ability to antagonize KDR signal activation. In oneembodiment, the acidic amino acid is E.

In another embodiment, the variant polypeptide of the invention can beany of the following: a polypeptide including SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:23, or SEQ ID NO:24; and a polypeptide having at least95% identity to a preceding polypeptide and having the ability toantagonize KDR signal activation.

The disease characterized by pathological angiogenesis may be cancer orocular disease. The ocular disease may be, macular degeneration, ordiabetic retinopathy. The cancer may be a metastatic cancer. Otherdiseases characterized by pathological angiogenesis are alsocontemplated and described elsewhere in this patent specification.

In another embodiment, the present invention includes a method forcreating a polypeptide capable of inhibiting angiogenesis whichcomprises the steps of providing a native VEGF comprising a variantC-terminal heparin binding domain and a native receptor tyrosine kinasebinding domain and modifying said native VEGF to form a variant VEGFpolypeptide. The variant VEGF polypeptide includes (1) SEQ ID NO: 25,SEQ ID NO: 26 or SEQ ID NO:27 where Xaa is an acidic amino acid; and (2)a polypeptide having at least 95% identity to a polypeptide of (1) andhaving the ability to antagonize KDR signal activation. In oneembodiment the polypeptide comprises a variant C-terminal heparindomain.

The present invention also includes polynucleotides. In one embodiment,the polynucleotides of the present invention include a polynucleotidethat encodes a vascular endothelial cell growth factor (VEGF) variantpolypeptide comprising a C-terminal heparin binding domain, wherein saidvariant polypeptide has one or more amino acid alternations from anative VEGF polypeptide, wherein said variant polypeptide binds heparinat a lower affinity than and/or repels heparin as compared to saidnative VEGF, and wherein the affinity of the variant polypeptide forboth VEGFR-1 (FLT-1) and VEGFR-2 (KDR/FLK-1) is substantially maintainedin comparison to said native VEGF, and wherein said variant polypeptideantagonizes KDR signal activation.

In one embodiment, the polynucleotide can include the followingpolynucleotides: a polynucleotide which encodes at least one of thefollowing polypeptides: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; and apolynucleotide having at least 95% identity to a preceding polypeptideand encoding a polypeptide having the ability to antagonize KDR signalactivation. In another embodiment, the polynucleotide can be any of thefollowing: a polynucleotide comprising, SEQ ID NO:5, SEQ ID NO:9, SEQ IDNO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, and SEQ ID NO:33; and a polynucleotide having atleast 95% identity any of the preceding polynucleotides and encoding apolypeptide having the ability to antagonize KDR signal activation.Other embodiments include those where the coding sequence found in SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33(base pairs 938-1513 in each SEQ ID) is replaced with base pairs1016-1513 of any of these SEQ ID's which correspond to the mature VEGFisoform or base pairs 1346-1513 of any of these SEQ IDs which correspondto the heparin binding domain. Embodiments having at least 95% sequenceidentity with these polynucleotides are also contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Conservation of primary HS binding residues in VEGF165 HSbinding domain.

FIG. 1A Amino acid sequences, in single letter code, of VEGF165 proteinprecursors from the vertebrate species and GenBank accession filesindicated at right. The multiple alignment was created using the BLASTMULTIALIGN tool (Altschul et al., 1990). Note that all GenBank sequencefiles of VEGF-A precursors include the signal peptide sequence asresidues 1-26, indicated by the box. Proteolytic cleavage removes thesignal peptide from mature VEGF-A proteins, yielding an amino-terminusindicated by the arrow. The names of various VEGF-A isoforms, such asVEGF165, VEGF189, etc., are derived from the number of amino acidresidues in the mature protein, after cleavage of the signal peptide.Thus the VEGF165 precursor contains 191 residues, but the mature VEGF165protein contains precursor residues 27-191, or 165. The GenBanknumbering scheme is not frequently used in the VEGF literature; instead,residue 27 of the precursor is most often referred to as residue 1. Theheparin binding domain (HBD) sequence is indicated by the box. Positionshighlighted in the HBD indicate primary HS binding sites as determinedpreviously (Krilleke et al., 2007).

FIG. 1B. Schematic diagram of Group 1 of mature VEGF165 proteinisoforms, which contain an amino-terminal VEGR receptor tyrosine kinase(RTK) binding region and a carboxy-terminal heparin binding domain(HBD). Numbering of amino acid residues in the mature VEGF165 proteinsof Group 1 is shown above the schematic; the amino acid sequence andcritical HS binding residues in the HBD are identified below, numberedrelative to their position in the HBD alone. For the group of VEGFAisoforms depicted in this Figure, identifying the critical HS bindingresidues as R13, R14 and R49 of the HBD provides an unambiguous anduniform means of identification in all VEGF-A heparin binding isoforms,independent of total protein length or HBD position.

FIG. 1C. FIG. 1C shows an alignment between heparin binding domains of asecond group, Group 2, which contains heparin binding domains withinsertions. As can be seen from both the alignment and the schematics,the HBD insertions in Group 2 change the numbering of the critical HSbinding residues (13,14,49 in Group 1) to 31,32,67 in Group 2a; 37,38,73in Group 2b; and 54,55,90 in Group 2c.

FIG. 2. VEGF165 HS binding domain structure. Models depicting the highresolution three dimensional structure of the VEGF165 HS binding domaindetermined previously by NMR spectrometry (Fairbrother et al., 1998;Zhou et al., 1998) (Protein Data Bank codes 2HGF and 1KMX). Models werecreated using PyMOL (The PyMOL Molecular Graphics System, Version 1.3,Schrödinger, LLC); the peptide backbone folding pattern (top and middlepanels) show amino terminal residues, carboxyl terminal residues, andthe positions (top panel) and side chains (middle panel) of primary HSbinding residues. Numbering of the critical HS binding residues is inthe context of the HBD alone, as detailed in FIG. 1B. The space fillmodel (lower panel) show the positive surface charge distribution ofprimary HS binding residues in.

FIG. 3. Human VEGF-A protein assay. Standard curve for theelectrochemiluminescent two-site immunoassay for VEGF-A: signalintensity (SI) vs. VEGF protein concentration.

FIG. 4. VEGF165 3S protein retains normal mass, immunoreactivity and KDRbinding but not signaling activity.

-   A. Mean VEGF165 protein content (ng/mg total cell protein+/−SD; n=3)    in 24 h conditioned media (clear bars) or low-volume detergent    extracts (gray bars) prepared from 293/KDR cells transfected with    plasmids encoding empty vector (empty), VEGF165 WT (WT), or VEGF165    3S (3S).-   B. VEGF165 3S protein (3S; left) in 24 h conditioned media prepared    from VEGF165 3S transfected 293/KDR cells visualized after SDS-PAGE    and immunoblotting. Three amounts (ng) of purified VEGF165 WT were    loaded on the right for reference (arrow).-   C. Saturation binding of KDR ectodomain-IgG fusion protein to    VEGF165 WT (squares) or VEGF165 3S proteins (circles) in vitro.    Values are mean KDR bound (ng/ml)+/−SD (n=3).-   D. Mean phospho-KDR level (signal intensity+/−SD; n=3) in 293/KDR    cells stably transfected with plasmids encoding empty vector (empty;    unfilled bar), VEGF165 WT (WT; light gray bar), VEGF165 3S (3S; dark    gray bar), or empty vector cells treated with purified VEGF165 WT    protein (2.5 nM) for 20 min (+VEGF; black bar).-   E. Growth rate (mean cell number+/−SD, n=3) of cultured 293/KDR    cells stably transfected with plasmids encoding empty vector    (circles), VEGF165 WT (squares), or VEGF165 3S (triangles).-   F. Soft agar colony formation by 239/KDR cells stably transfected    with empty vector, (left), VEGF 3S expression plasmid (middle), or    VEGF WT expression plasmid (right).

FIG. 5. Competitive antagonism of VEGFR activation and VEGFR-drivenanchorage independent cell growth and tumorigenesis by VEGF165 3Sprotein.

-   A. VEGF protein content in media conditioned by 293/KDR cells    transfected with VEGF165 3S plasmid (left) or empty plasmid (right),    before (“none”) or after immunodepletion (ID) using anti-VEGF-A    (“−VEGF”) or an unrelated control antibody (“mock”), expressed as    mean ng/mg total protein+/−SD (n=3).-   B. Phospho-KDR levels (% maximum, +/−SD, n=3) in serum-deprived    293/KDR cells treated with VEGF165 WT protein (10 ng/ml) in the    presence of concentrated conditioned media from 293/KDR VEGF 3S    transfectants that had been immunodepleted using a non-specific    control antibody (triangles) or anti-VEGF (circles). The x-axis    indicates the concentration of VEGF165 3S protein (ng/ml) in the    conditioned media prior to immunodepletion.-   C. Soft agar colony formation by untreated 239/KDR cells stably    transfected with an expression plasmid for VEGF165 WT (upper left    panel), cells treated with the indicated concentrations of the VEGR    inhibitor pazopanib (upper middle and right panels), or cells    treated with conditioned medium containing the indicated    concentrations of VEGF165 3S protein (lower panels).-   D. Mean tumor volume (mm3+/−SD) in mice (n=5/group) implanted with    293/KDR cells (3×10⁶ cells per animal) stably expressing VEGF165 3S    (inverted triangles), VEGF165 WT (squares), or empty vector    (circles), measured at the indicated times post-implantation. Other    groups were implanted with a mixture of empty vector cells and    VEGF165 WT transfectants at 1.5×10⁶ cells each (triangles), or a    mixture of VEGF165 WT and VEGF165 3S transfectants at 1.5×10⁶ cells    each (diamonds).

FIG. 6. Nucleotide alignments of thirteen human VEGF alpha sequences'HBD. The 6 most abundant in nature are Group 1. 7 rarer transcriptscontain HBDs with insertions, and are called Group 2.

FIG. 7. VEGF165 3S and WT both form disulfide-linked protein dimers ofthe anticipated molecular mass.

FIG. 8. VEGF165 3S is not oligomerized by heparin sulfate.

FIG. 9A-C. VEGF165 3S antagonism follows the pattern of VEGFA/VEGFRbinding interaction.

FIG. 10A-B. VEGF165 3S antagonism is independent of NRP1 interaction.

DETAILED DESCRIPTION

In the present invention, the inventors found that when HSP binding toresidues R123, R124, and R159 of VEGF 165 was disrupted using combinedopposite charge substitutions, unexpectedly and surprisingly theresultant polypeptide was a competitive antagonist of VEGF signaling andangiogenesis. The inventors' results suggest that receptor occupancycombined with charge-based repulsion of HSP from the ligand/receptorcomplex constitutes an effective general strategy for antagonizing VEGFsignaling.

The inventors generated a cDNA to encode opposite charge substitutionsat three critical HS binding residues in human VEGF165, positionsR123E/R124E/R159E in the mature VEGF165 protein, numbered from the siteof signal peptide cleavage (designated VEGF165 3S; FIGS. 1A and B). Forconvenience in identifying these residues in the context of other VEGF-Aisoforms that also contain the heparin binding domain (HBD; the HBDsequence is identical in for some VEGF-A isoforms, but the HBD for otherisoforms have insertions/deletions; see the list of VEGF-A isoformsequences discussed elsewhere in the Specification, shown at FIG. 1B,1C, and FIG. 6). In the group of HBD referred to as Group 1, thepositions of these residues relative to the heparin binding domain alonemay be referred to as R13E, R14E and R49E, as depicted in FIG. 1B. Theseare depicted in the three dimensional NMR structure of the VEGF165heparin binding domain in FIG. 2. FIG. 1C shows that there are also HBDsthat have insertions/deletions in the HBD that change the relativenumbering of the positions of these residues in the HBD domain, asdepicted in the figure and in FIG. 6. Expression plasmids encodingvector alone, VEGF165 WT, or VEGF165 3S were stably transfected intoHEK293 cells that had been engineered previously to express 2.5×10⁶VEGFR2 per cell (293/KDR; Backer et al., 2005). The inventors hadanticipated that VEGF165 WT expression in these cells would result inautocrine KDR activation and cell transformation. The inventors hadexpected VEGF165 3S to lack KDR activating ability and thus to have noeffect on phenotype. Ectopic VEGF protein production by transfectantswas measured using a two-site immunoassay with a detection limit of 37.5attomoles/25 ul (1.5 pM) VEGF (FIG. 3). Among marker selected masscultures, WT transfectants produced ˜1.0 ng/ml/24 h VEGF165 protein inconditioned media, 3S transfectants produced ˜2.5 ng/ml/24 h, and VEGFprotein was undetectable in the empty vector control media (FIG. 4A,white bars). The VEGF content in low volume detergent extracts from thesame cells was proportionally higher, as expected (FIG. 4A, gray bars).Molecular mass and antibody recognition of VEGF165 3S protein inconditioned media were indistinguishable from WT (FIG. 4B). Saturationbinding of VEGF165 3S to KDR in vitro (FIG. 4C, squares; KD ˜19 pM) wasalso equivalent to VEGF165 WT binding (FIG. 4C, circles; KD ˜23 pM) andconsistent with published steady-state binding affinity values (Ferrara,2004).

Thus, the inventors found that while VEGF165 3S binds KDR like VEGF165WT protein, the mutant failed to induce KDR kinase activity andantagonized VEGF165 WT binding and signaling in vitro and in vivo. Theseresults show a critical role for HS in receptor activation and show thattargeted disruption of critical HS binding sites is a feasibleantagonist development strategy for VEGF. This work shows that thatcharge based repulsion of HSP from the ligand/receptor complex is aneffective general strategy for antagonizing signaling by VEGF.

These results are surprising in view of previous work where theseresidues on VEGF165, mutated from arginine to alanine, resulted in amutant that retained full activity as an agonist in stimulatingangiogenesis.

Without being bound by theory, the inventors' understanding of KDRactivation by VEGF165 and HS suggests a mechanistic basis for signalingantagonism by the targeted disruption of HS-ligand binding. The VEGFbinding site in KDR encompasses IgG-like domains (D) 1-3: D2 containsprimary contacts and D1 and D3 contribute to overall binding affinityand specificity. HS was found to be required for high affinity bindingof VEGF165 to KDR ectopically expressed in HS-negative CHO pgsA 745cells. Similar to Met and fibroblast growth factor receptors, KDR alsointeracts directly with HS, through at least one site located between D6and D7. Recent structural studies of the KIT, PDGF and VEGF receptorfamilies highlight the importance of homotypic receptor-receptorinteraction domains in stabilizing receptor dimerization and kinasetransactivation: direct contacts between D4 domains for KIT and D7domains for KDR are enabled by ligand-induced conformational changes inthe receptor ectodomain. In these models, sequential binding eventspromote and incrementally stabilize HS-ligand-receptor aggregatescapable of signaling. Without being bound by theory, the inventorshypothesize that VEGF165 first binds KDR D2; the weaker VEGF165-D1 and-D3 interactions may be stabilized by HS-VEGF interaction and byHS-VEGF-KDR bridging at D6/D7. VEGF-HS also may also induceconformational changes that enable the apposition of KDR D7 domains andin turn, homotypic interaction and kinase transactivation. Ourobservations underscore the importance of specific HS interactions infacilitating events after ligand binding that are required for KDRactivation, and expose their susceptibility to targeted disruption.

HS binding growth factors promote tumor growth, angiogenesis andmetastasis in a variety of human cancers. In addition to the strategydescribed here, oligosaccharide HS mimetics and modified heparinfragments can act as HS binding antagonists. However, the complexity ofHS glycosaminoglycans (GAGs) challenges the development of potent andselective agents, and frequent alterations in HS GAG composition intumors may render such agents less competitive for ligand binding. Incontrast, opposite substitutions to critical HS binding residues in anotherwise wild type protein ligand are simple to introduce, unaffectedby tumor HS GAG composition, and inherently pathway selective. Thesefeatures suggest that the inventors' approach is an expedient andpractical route for the development of antagonists of HS binding growthfactors, in particular, VEGF165.

In one aspect, the present invention provides novel VEGF variants. Inparticular, the invention relates to variants of vascular endothelialgrowth factor-A (VEGF-A). VEGF-A is an essential regulator of normalangiogenesis during embryogenesis and adulthood, and participates inpathological angiogenesis in many diseases, including many prevalentforms of cancer. As is known in the art, VEGF-A signaling is mediatedthrough the cell surface receptor tyrosine kinases VEGFR1 (Flt-1) andVEGFR2 (Flk-1, KDR). Differential splicing of VEGF-A pre-mRNA results inthe expression of multiple VEGF-A isoforms, the most abundant of whichin humans are VEGF121, VEGF 165 and VEGF 189. All isoforms contain thesame binding sites for VEGFR1 and VEGFR2, but differ in their capacityto bind to heparan sulfate proteoglycans (HSPs) ubiquitously present inthe extracellular matrix and on target cell surfaces, due to thepresence or absence of two basic heparin-binding domains (HBDs) encodedby exons 6 and 7. VEGF 121 lacks a HBD and is freely diffusible in theextracellular space, whereas VEGF 165 (SEQ ID NO:3) and VEGF 189 displaymoderate and high affinity HSP binding, respectively, limiting themobility of these isoforms in HSP-rich compartments. The exon 7-encodedpeptide also contains residues that enable VEGF 165 to bind to the VEGFco-receptor NRP1.

In one embodiment, the present invention relates to variants of VEGF165. SEQ ID NO:2 shows vascular endothelial growth factor A isoform 1precursor from Homo sapiens. This form is 191 amino acids in length. Asignal sequence is cleaved off to form the mature vascular endothelialgrowth factor A isoform 1 Homo sapiens SEQ ID NO:3. SEQ ID NO:3 may bereferred to as “VEGF 165” and is 165 amino acids in length. VEGF 165 isalso referred to herein and elsewhere as native VEGF 165, mature VEGF165, and the like. SEQ ID NO:4 is the carboxyl terminal region of SEQ IDNO:2 and SEQ ID NO:3, and corresponds to residues 111-165 of SEQ ID NO:3and is defined by a plasmin cleavage site. Together, SEQ ID NO:2-4 canbe referred to herein as VEGF 165 sequences; they may also be referredto as “native VEGF amino acid sequences” or “native VEGF” or “nativeVEGF polypeptides”. They may also be referred to as “native VEGF 165amino acid sequences” or “native VEGF 165” or “native VEGF 165polypeptides”.

The VEGF variants described herein represent VEGF 165 sequences thathave been modified in accordance with the invention. These variantscomprise at least one polypeptide having a modified heparin bindingdomain. These polypeptides may be referred to as VEGF variantpolypeptides, a VEGF variant amino acid sequence, VEGF 165 variants,VEGF 165 variant amino acid sequences, VEGF 165 variant polypeptides,and the like. The VEGF variants described herein may also be referred toas “polypeptides of the present invention” or “VEGF variants of thepresent invention,” “VEGF sequences of the present invention,” and thelike.

A “native sequence” protein comprises the amino acid sequence of aprotein as found in nature, e.g. in a human. The native sequence proteincan be made by recombinant or other synthetic means, or may be isolatedfrom a native source.

The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-α-amino acids. This definition is meant to includenorleucine, ornithine, and homocysteine. The amino acids are identifiedby either the single-letter or three-letter designations, as follows:Asp (D) aspartic acid Thr (T) threonine Ser (S) serine Glu (E) glutamicacid Pro (P) proline Gly (G) glycine Ala (A) alanine Cys (C) cysteineVal (V) valine Met (M) methionine Ile (I) isoleucine Leu (L) leucine Tyr(Y) tyrosine Phe (F) phenylalanine His (H) histidine Lys (K) lysine Arg(R) arginine Trp (W) tryptophan Gln (Q) glutamine Asn (N) asparagine.

The present invention, in part, relates to VEGF variants having one ormore substitutions on the heparin binding domain of negatively charged(e.g., acidic) amino acid residues for amino acid residue arginine atcertain specified positions on a VEGF polypeptide, as disclosed herein.Appropriate VEGF polypeptides with which to create variants arediscussed herein and including human alternately spliced VEGF isoformsof 121, 145, 165, 183, 189, and 206 amino acids in length, and speciesorthologs of VEGF. Specific arginine residues to substitute with anacidic amino acid, for a specific VEGF polypeptide, are disclosedherein. An acidic amino acid is defined herein as an amino acid residuehaving a negative charge at physiological pH. In one embodiment, anacidic amino acid is aspartic acid (Asp, D) or glutamic acid (Glu, E).In one embodiment, an acidic amino acid is glutamic acid. Heparinsulfate (HS) binding is a charge-based interaction wherein sulfatemoieties in HS bind to basic residues (R or K) in the VEGF HS bindingdomain. Changing the basic R residues or K residues to either D residuesor E residues has the same impact-repulsion of the HS sulfate moietiesfrom the VEGF-VEGFR complex, and signaling antagonism.

In one embodiment, the heparin binding domain of a native VEGF 165sequence of the present invention is modified by substituting negativelycharged (e.g., acidic) amino acid residues for amino acid residuearginine at certain specified positions on a VEGF polypeptide. VEGFvariant polypeptides of the invention include SEQ ID NO:25, SEQ IDNO:26, or SEQ ID NO:27. SEQ ID NO:25 is the same as SEQ ID NO:2, exceptthat one or more of positions 149, 150, and 185 of SEQ ID NO:25 cancontain an acidic amino acid residue in place of the arginine atcorresponding positions of SEQ ID NO:2. SEQ ID NO:26 is the same as SEQID NO:3, except that one or more of positions 123, 124, and 159 of SEQID NO:26 can contain an acidic amino acid in place of the arginine atcorresponding positions of SEQ ID NO:3. SEQ ID NO:27 is the same as SEQID NO:4, except that one or more of positions 13, 14, and 49 of SEQ IDNO:27 can contain an acidic amino acid in place of arginine atcorresponding positions of SEQ ID NO:4.

In another embodiment, the heparin binding domain is modified bysubstituting negatively charged (e.g., acidic) amino acid residues foramino acid residue arginine at certain specified positions on a VEGFpolypeptide. VEGF variant polypeptides of the invention include SEQ IDNO:6, SEQ ID NO:7, or SEQ ID NO:8. SEQ ID NO:6 is the same as SEQ IDNO:2, except that positions 149, 150, and 185 of SEQ ID NO:6 contain aglutamic acid in place of the arginine at corresponding positions of SEQID NO:2. SEQ ID NO:7 is the same as SEQ ID NO:3, except that atpositions 123, 124, and 159 of SEQ ID NO:7 contain a glutamic acid inplace of the arginine at corresponding positions of SEQ ID NO:3. SEQ IDNO:7 is also referred to herein as “3S VEGF 165.” SEQ ID NO:8 is thesame as SEQ ID NO:4, except that at positions 13, 14, and 49 of SEQ IDNO:8 contain a glutamic acid in place of arginine at correspondingpositions of SEQ ID NO:4.

Other VEGF variant polypeptides of the invention include SEQ ID NO:10,SEQ ID NO:11, or SEQ ID NO:12. SEQ ID NO: 10 is the same as SEQ ID NO:2,except that positions 149 and 150 of SEQ ID NO:10 contain a glutamicacid in place of the arginine at corresponding positions of SEQ ID NO:2.SEQ ID NO:11 is the same as SEQ ID NO:3 except that positions 123 and124 of SEQ ID NO:11 contain a glutamic acid in place of the arginine atcorresponding positions of SEQ ID NO:3. SEQ ID NO:12 is the same as SEQID NO:4, except that positions 13 and 14 of SEQ ID NO:12 contain aglutamic acid in place of arginine at corresponding positions of SEQ IDNO:4.

Other VEGF variant polypeptides of the invention include SEQ ID NO:14,SEQ ID NO:15, or SEQ ID NO:16. SEQ ID NO: 14 is the same as SEQ ID NO:2,except that position 185 of SEQ ID NO:14 contains a glutamic acid inplace of the arginine at the corresponding position of SEQ ID NO:2. SEQID NO:15 is the same as SEQ ID NO:3 except that position 159 of SEQ IDNO:15 contains a glutamic acid in place of the arginine at thecorresponding position of SEQ ID NO:3. SEQ ID NO:16 is the same as SEQID NO:4, except that position 49 of SEQ ID NO:16 contains a glutamicacid in place of arginine at the corresponding position of SEQ ID NO:4.

Other VEGF variant polypeptides of the invention include SEQ ID NO:18,SEQ ID NO:19, or SEQ ID NO:20. SEQ ID NO: 18 is the same as SEQ ID NO:2,except that position 150 of SEQ ID NO:14 contains a glutamic acid inplace of the arginine at the corresponding position of SEQ ID NO:2. SEQID NO:19 is the same as SEQ ID NO:3 except that position 124 of SEQ IDNO:19 contains a glutamic acid in place of the arginine at thecorresponding position of SEQ ID NO:3. SEQ ID NO:20 is the same as SEQID NO:4, except that at position 14 of SEQ ID NO:20 contains a glutamicacid in place of arginine at the corresponding position of SEQ ID NO:4.

Other VEGF variant polypeptides of the invention include SEQ ID NO:22,SEQ ID NO:23, or SEQ ID NO:24. SEQ ID NO: 22 is the same as SEQ ID NO:2,except that position 149 of SEQ ID NO:22 contains a glutamic acid inplace of the arginine at the corresponding position of SEQ ID NO:2. SEQID NO:23 is the same as SEQ ID NO:3 except that position 123 of SEQ IDNO:23 contains a glutamic acid in place of the arginine at thecorresponding position of SEQ ID NO:3. SEQ ID NO:24 is the same as SEQID NO:4, except that at position 13 of SEQ ID NO:24 contains a glutamicacid in place of arginine at the corresponding position of SEQ ID NO:4.

In one embodiment, the variant vascular endothelial cell growth factor(VEGF) variant polypeptide comprises a variant C-terminal heparinbinding domain and a native receptor tyrosine kinase binding domain,wherein said variant polypeptide has one or more amino acid alternationsfrom a native VEGF polypeptide, wherein said variant polypeptide bindsheparin at a lower affinity than and/or repels heparin as compared tosaid native VEGF, and wherein the affinity of the variant polypeptidefor both VEGFR-1 (FLT-1) and VEGFR-2 (KDR/FLK-1) is substantiallymaintained in comparison to said native VEGF amino acid sequence, andwherein said variant polypeptide antagonizes KDR signal activation. Inone embodiment the polypeptide comprises a variant C-terminal heparindomain.

In one embodiment, a native VEGF polypeptide refers to one or more ofthe polypeptides comprising SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

In one embodiment, a variant VEGF polypeptide comprises a polypeptidewhich comprises SEQ ID NO:25 wherein the amino acid of at least one ofpositions 149, 150, and 185 of SEQ ID NO:25 is an acidic amino acid. Anacidic amino acid is defined herein as an amino acid residue having anegative charge at physiological pH. In one embodiment, an acidic aminoacid is aspartic acid (Asp, D) or glutamic acid (Glu, E). In anotherembodiment, a variant VEGF polypeptide comprises a polypeptide whichcomprises SEQ ID NO:26 wherein the amino acid of at least one ofpositions 123, 124, and 159 of SEQ ID NO:26 is an acidic amino acid. Inanother embodiment, a variant VEGF polypeptide comprises a polypeptidewhich comprises SEQ ID NO:27 wherein the amino acid of at least one ofpositions 13, 14, and 49 of SEQ ID NO:27 is an acidic amino acid.

In another embodiment, the present invention includes VEGF189 variantpolypeptides and polynucleotides. VEGF189 variant polypeptides andpolynucleotides are differential splicing isoforms from VEGF165. VEGF189variant polypeptides of the invention include VEGF189 which have acidicamino acid changes at one or more of the equivalent (conserved) sites asthe ones described for VEGF165. On the mature form of VEGF189, these areR147, 148, and 183. VEGF 189 display moderate and high affinity HSPbinding, respectively, limiting the mobility of these isoforms inHSP-rich compartments. VEGF189 variant polypeptides of the inventioninclude the following polypeptides: VEGF 189 precursor of 215 aminoacids having E at positions 173, 174, and 209 (SEQ ID NO:34), VEGF 189precursor of 215 amino acids having E at positions 173, 174 (SEQ IDNO:35), VEGF 189 precursor of 215 amino acids having E at position 209(SEQ ID NO:36), VEGF 189 precursor of 215 amino acids having E atposition 174 (SEQ ID NO:37), and VEGF 189 precursor of 215 amino acidshaving E at position 173 (SEQ ID NO:38). In SEQ ID NOs: 34 through 38the alternations to obtain the variant VEGF189 are found at one or moreof AA 173, 174 and 209. The invention includes the mature forms of theforegoing amino acids of SEQ ID NO:34, 35, 36, 37, and 38. The inventionalso includes polynucleotides that include polynucleotides that encodeany of the VEGF189 variant polypeptides of the invention.

Thus, the present invention includes polypeptides wherein the variantpolypeptide includes the following: a polypeptide comprising the matureform of a polypeptide which include the following: SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; and a polypeptidehaving at least 95% identity to a polypeptide described above, andhaving the ability to antagonize KDR signal activation.

Other variant VEGF polypeptides of the present invention include otherVEGF-A isoforms comprising a C-terminal domain containing a heparinbinding domain comprising the amino acid substitutions of the presentinvention. Such GenBank Files containing VEGF-A isoforms that containthe heparin binding domain which can be modified in accordance with thepresent invention. e.g., having a C terminal domain containing a heparinbinding domain comprising the amino acid substitutions described herein.These include vascular endothelial growth factor A isoform o precursor(Homo sapiens) which have modifications at equivalent positions on theamino acid sequence as the other variant sequences claimed herein. Asdiscussed herein at FIG. 1B, Group 1 sequences for the isoformsdisclosed in FIG. 1B have modifications in the HBD of 13, 14, and 49positions to an acidic amino acid, wherein in one embodiment, the acidicamino acid is E. Group 1 sequences include NP_(—)001165097.1;NP_(—)001165100.1; NP_(—)0010289928.1, NP_(—)001020539, BAG70265.1,BAG70136.1. NP_(—)001165100.1 GI:284172471; vascular endothelial growthfactor A isoform 1 precursor (Homo sapiens) is represented herein as SEQID NO:47, with a C terminal domain (HBD) of SEQ ID NO:68.NP_(—)001165097.1, GI:284172465; is SEQ ID NO:48, with a C terminaldomain (HBD) of SEQ ID NO:4. Vascular endothelial growth factor Aisoform k precursor (Homo sapiens), a 209 aa protein, NP_(—)001165096.1,GI:284172463; is represented herein as represented herein as SEQ IDNO:49, with a C terminal domain (HBD) of SEQ ID NO:64. Vascularendothelial growth factor A isoform j precursor (Homo sapiens), a 215 aaprotein, NP_(—)001165095.1, GI:284172461 is represented herein as SEQ IDNO:50, with a C terminal domain (HBD) of SEQ ID NO:65. Vascularendothelial growth factor A isoform i precursor (Homo sapiens), a 232 aaprotein, NP_(—)001165094.1GI:284172459 is represented herein as SEQ IDNO:51,with a C terminal domain (HBD) of SEQ ID NO:67. Vascularendothelial growth factor A isoform g (Homo sapiens), a 371 aa protein,NP_(—)001028928.1GI:76781487 is represented herein as SEQ ID NO:52, witha C terminal domain (HBD) of SEQ ID NO:68. Vascular endothelial growthfactor A isoform d (Homo sapiens), a 371 aa protein, NP_(—)001020539.2,GI:76781483; is represented herein as SEQ ID NO:53, with a C terminaldomain (HBD) of SEQ ID NO:4. Vascular endothelial growth factor Aisoform c (Homo sapiens) a 389 aa protein, NP_(—)001020538.2,GI:76781482, is represented herein as SEQ ID NO:54, with a C terminaldomain (HBD) of SEQ ID NO:64. Vascular endothelial growth factor Aisoform b (Homo sapiens) a 395 aa protein, NP_(—)003367.4, GI:76781481is represented herein as SEQ ID NO:55, with a C terminal domain (HBD) ofSEQ ID NO:65. Vascular endothelial growth factor A isoform a (Homosapiens), a 412 aa protein, NP_(—)001020537.2, GI:76781480, isrepresented herein as SEQ ID NO:56, with a C terminal domain (HBD) ofSEQ ID NO:67. Vascular endothelial growth factor isoform VEGF165 (Homosapiens), a 191 aa protein, BAG70265.1, GI:197692603, is representedherein as SEQ ID NO:57, with a C terminal domain (HBD) of SEQ ID NO:4.Vascular endothelial growth factor isoform VEGF165 (Homo sapiens), a 191aa protein, BAG70136.1GI:197692345 , is represented herein as SEQ IDNO:58, with a C terminal domain (HBD) of SEQ ID NO:4. Vascularpermeability factor, a 232 aa protein P15692.2, GI:17380528, isrepresented herein as SEQ ID NO:59, with a C terminal domain (HBD) ofSEQ ID NO:67. See FIG. 6, showing an alignment of thirteen human VEGFalpha sequences (isoforms) heparin binding domains. The six mostabundant isoforms in nature are grouped as Group 1 (see FIG. 1B). FIG.1C shows an alignment between heparin binding domains of a second group,Group 2, which contains heparin binding domains with insertions. As canbe seen from both the alignment and the schematics, the HBD insertionsin Group 2 change the numbering of the critical HS binding residues(13,14,49 in Group 1) to 31,32,67 in Group 2a; 37,38,73 in Group 2b; and54,55,90 in Group 2c.

Thus, variant polypeptides of the invention include polypeptidescomprising a polypeptide which include the following: SEQ ID NO:4,wherein the amino acid of at least one of positions 13, 14, and 49 ofthe sequence of SEQ ID NO:4 is an acidic amino acid; SEQ ID NO:64,wherein the amino acid of at least one of positions 31, 32, and 67 ofthe sequence of SEQ ID NO:64 is an acidic amino acid; SEQ ID NO:65,wherein the amino acid of at least one of positions 37, 38, and 73 ofthe sequence of SEQ ID NO:65 is an acidic amino acid, SEQ ID NO:67,wherein the amino acid of at least one of positions 54, 55, and 90 ofthe sequence of SEQ ID NO:67 is an acidic amino acid, and SEQ ID NO:68,wherein the amino acid of at least one of positions 13, 14, and 49 ofthe sequence of SEQ ID NO:68 is an acidic amino acid; as well aspolypeptides having at least 95% identity to a polypeptide describedabove, and having the ability to antagonize KDR signal activation.

Variant polypeptides of the invention also include a polypeptidecomprising a polypeptide which include the following: SEQ ID NO:47,wherein the amino acid of at least one of positions 149, 150 and 185 ofSEQ ID NO:47 is an acidic amino acid, SEQ ID NO:48, wherein the aminoacid of at least one of positions 149, 150, and 185 of SEQ ID NO:48 isan acidic amino acid, SEQ ID NO:49, wherein the amino acid of at leastone of positions 167, 168 and 203 of SEQ ID NO:49 is an acidic aminoacid, SEQ ID NO:50, wherein the amino acid of at least one of positions173, 174, and 209 of SEQ ID NO:50 is an acidic amino acid, SEQ ID NO:51,wherein the amino acid of at least one of positions 190, 191, and 226 ofSEQ ID NO:51 is an acidic amino acid, SEQ ID NO:52, wherein the aminoacid of at least one of positions 329, 330, and 365 of SEQ ID NO:52 isan acidic amino acid, SEQ ID NO:53, wherein the amino acid of at leastone of positions 329, 330, and 365 of SEQ ID NO:53 is an acidic aminoacid, SEQ ID NO:54, wherein the amino acid of at least one of positions347, 348, and 383 of SEQ ID NO:54 is an acidic amino acid, SEQ ID NO:55,wherein the amino acid of at least one of positions 353, 354, and 389 ofSEQ ID NO:55 is an acidic amino acid, SEQ ID NO:56, wherein the aminoacid of at least one of positions 370, 371 and 406 of SEQ ID NO:56 is anacidic amino acid, SEQ ID NO:57, wherein the amino acid of at least oneof positions 149, 150 and 185 of SEQ ID NO:57 is an acidic amino acid,SEQ ID NO:58, wherein the amino acid of at least one of positions 149,150 and 185 of SEQ ID NO:58 is an acidic amino acid, and SEQ ID NO:59,wherein the amino acid of at least one of positions 190, 191, and 226 ofSEQ ID NO:59 is an acidic amino acid; as well as polypeptides having atleast 95% identity to a polypeptide described above, and having theability to antagonize KDR signal activation.

The present invention, then, also includes a vascular endothelial cellgrowth factor (VEGF) variant polypeptide comprising a C-terminal heparinbinding domain, wherein said variant polypeptide has one or more aminoacid alternations from a native VEGF polypeptide, where the variantincludes a heparin sulfate binding domain (HBD) wherein HBD can comprisea HBD as described in FIG. 1B or FIG. 1C or FIG. 6. For example, the HBDcan contain a splice site as shown in FIG. 1C and FIG. 6 with additionalsequences spliced in, thus adjusting the position of the variantresidues based on a numbering position from position 1 in the HBD, asseen in FIG. 1C. For example, the HBD can comprise the sequence as shownin, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and/or SEQID NO:68, which have an insertion between residues 4 (E) and 6 (P) ofbetween 18-41 amino acid residues replacing residue 5(N) as shown inFIG. 6, wherein at least one of equivalent of positions 13, 14, and 49as shown in SEQ ID NO:4 is substituted with an acidic amino acid, suchas E or D. For example, for SEQ ID NO:64, the substitutions may occur atpositions 31, 32, and 67. For example, for SEQ ID NO:65, thesubstitutions may occur at positions 37, 38 and 73. For example, for SEQID NO:67, the substitutions may occur at positions 54, 55, and 90. ForSEQ ID NO:68, the variant positions are the same, but the C terminus hasa variant ending as shown in the FIG. 6. Specifically, the inserts caninclude

-   KKSVRGKGKGQKRKRKKSR (SEQ ID NO:60), see full length C terminal    domain SEQ ID NO:64; KKSVRGKGKGQKRKRKKSRYKSWSV (SEQ ID NO:61), see    full length C terminal domain SEQ ID NO:65,-   KKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPG (SEQ ID NO:62), see full    length C terminal domain SEQ ID NO:66,-   and KKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPH (SEQ ID NO:63), see    full length C terminal domain SEQ ID NO:67. The C terminus of the    HBD may vary with a SLTRKD (SEQ ID NO:69), see full length C    terminal domain SEQ ID NO:68.

For convenience these sequences may be called Group 2 sequences and thepositioning of the variant residues may be at residue 31, 31 and/or 67of the HBD for NP_(—)001020538.2 (SEQ ID NO:64) and NP_(—)001165096.1(SEQ ID NO:64), at residues 37, 38 and/or 73 of the HBD forNP_(—)001165095.1 (SEQ ID NO:65) and NP_(—)003367.4 (SEQ ID NO:65), andat residues 54, 55 and 90 of HBD for NP_(—)001020537.2 (SEQ ID NO:67),p15692.2(SEQ ID NO:67), and NP_(—)001165094.1(SEQ ID NO:67). The VEGFvariants of the present invention show at least about 75%, at leastabout 80%, at least about 85%, at least about 85%, at least about 86%,at least about 87%, at least about 88%, at least about 89%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99%, amino acidsequence identity with a polypeptide of the present invention, andoptionally having the ability to antagonize KDR signal activation.

Alternative embodiments within the scope of the invention include thosewhere the vascular endothelial cell growth factor (VEGF) variantpolypeptide may include amino acid residues as shown in FIG. 1A, 1B, 1C,and/or FIG. 6, among others, and include therefore isoforms of VEGFwhich contain an insertion in the heparin binding domain (HBD) at aminoacids 18-41 (see FIG. 1C and FIG. 6) as well as those naturallyoccurring VEGF isoforms which each contain the variants in the heparinbinding domain at the locations noted (see FIG. 1B). The VEGF variantpolypeptide has, in the case of molecules described in FIG. 1B, at leastone of the arginines at amino acids 13, 14 and/or 49 substituted with anegatively charged (e.g., acidic) amino acid residue, in someembodiments glutamic acid, or in the case of molecules described in FIG.1C, at least one of the arginines at amino acid 31, 32 and/or 67; 37, 38and/or 73; or 54, 55 and/or 90 substituted with a negatively charged(e.g., acidic) amino acid residue, in some embodiments glutamic acid.

The present invention also includes methods for treating a diseasecharacterized by pathological angiogenesis, comprising administering toa patient in need thereof a pharmaceutically effective amount of avascular endothelial cell growth factor (VEGF) variant polypeptidecomprising a variant C-terminal heparin binding domain, and a nativereceptor tyrosine kinase binding domain, wherein said variantpolypeptide has one or more amino acid alternations from a native VEGFpolypeptide, wherein said variant polypeptide binds heparin at a loweraffinity than and/or repels heparin as compared to said native VEGF. Inone embodiment the polypeptide comprises a variant C-terminal heparindomain.

As used herein, an “effective amount” or a “pharmaceutically effectiveamount” is an amount sufficient to effect beneficial or desired clinicalresults. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofa VEGF variant is an amount that is sufficient to palliate, ameliorate,stabilize, reverse, slow or delay the progression of the disease state.In a preferred embodiment of the invention, the “effective amount” isdefined as an amount capable of reducing the growth and/or remodeling ofcollateral blood vessels.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired clinical results. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment “Treatment” refers toboth therapeutic treatment and prophylactic or preventative measures.Those in need of treatment include those already with the disorder aswell as those in which the disorder is to be prevented. “Palliating” adisease means that the extent and/or undesirable clinical manifestationsof a disease state are lessened and/or the time course of theprogression is slowed or lengthened, as compared to a situation withouttreatment.

In one embodiment, the affinity of the variant polypeptide for bothVEGFR-1 (FLT-1) and VEGFR-2 (KDR/FLK-1) is substantially maintained incomparison to said native VEGF. In another embodiment, the variantpolypeptide antagonizes KDR signal activation.

“Angiogenesis” is defined the promotion of the growth of new bloodcapillary vessels from existing endothelium. The contribution ofelevated VEGF expression and its signaling pathway has been associatedto pathological angiogenesis in virtually all carcinomas studied as wellas neovascular ophthalmic diseases such as age-related maculardegeneration and retinopathy. Polypeptides of the invention may be usedto treat these disorders.

A disease characterized by pathological angiogenesis includes anydisease or pathological process that is characterized by undesirablegrowth of new blood vessels in the body. Thus the polypeptides of thepresent invention may be used to treat pathological conditions such astumor growth and in non-neoplastic diseases involving abnormalneovascularization. Accordingly examples of disorders characterized bypathological angiogenesis that are treatable by the present inventioninclude, but are not limited to, neoplastic diseases, including but notlimited to solid tumors, and excessive angiogenesis, involving, forexample, vascularization and/or inflammation, such as atherosclerosis,rheumatoid arthritis (RA), neovascular glaucoma, proliferativeretinopathy including proliferative diabetic retinopathy, maculardegeneration, age-related macular degeneration, wet maculardegeneration, hemangiomas, angiofibromas, and psoriasis. Othernon-limiting examples of non-neoplastic angiogenic disease areretinopathy of prematurity (retrolental fibroplastic), corneal graftrejection, insulin-dependent diabetes mellitus, multiple sclerosis,myasthenia gravis, Crohn's disease, autoimmune nephritis, primarybiliary cirrhosis, acute pancreatitis, allograph rejection, allergicinflammation, contact dermatitis and delayed hypersensitivity reactions,inflammatory bowel disease, septic shock, osteoporosis, osteoarthritis,cognition defects induced by neuronal inflammation, Osler-Webersyndrome, restinosis, and fungal, parasitic and viral infections,including cytomegaloviral infections. Conditions or diseases to whichpersistent or uncontrolled angiogenesis contribute have been termedangiogenic dependent or angiogenic associated diseases.

Neoplastic diseases to treat include cancer and/or metastatic cancer. Apartial list of cancers to treat with polypeptides of the presentinvention include solid tumors, such as carcinomas, derived fromepithelial cells, including breast, prostate, lung, pancreas, and colon;sarcomas, arising from connective tissue, such as bone cancer; lymphomaand leukemias, which arise from hematopoietic cells; germ cell tumorssuch as testicular cancer and ovarian cancer, and blastomas, derivedfrom precursor or embryonic tissue. Such tumors include benign ormalignant tumors (e.g. renal, liver, kidney, bladder, breast, gastric,ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid,hepatic carcinomas; sarcomas; glioblastomas; and various head and necktumors); leukemias and lymphoid malignancies; other disorders such asneuronal, glial, astrocytal, hypothalamic and other glandular,macrophagal, epithelial, stromal and blastocoelic disorders; andinflammatory, angiogenic and immunologic disorders.

The identification of such disease is well within the ability andknowledge of one skilled in the art. For example, human individuals whoare either suffering from a clinically significant neoplastic orangiogenic disease or who are at risk of developing clinicallysignificant symptoms are suitable for administration of the present VEGFreceptor antibodies. A clinician skilled in the art can readilydetermine, for example, by the use of clinical tests, physicalexamination and medical/family history, if an individual is a candidatefor such treatment.

Compositions of the present invention may also be useful as molecularimaging agents for VEGFR. Compositions of the present invention may beused to bind to the target molecule (VEGFR, for example) and labeledwith a moiety that renders it visible to a particular imaging modality.Labels in common use include radionuclides, fluorescent molecules, andparamagnetic ions, as well as nanoparticles, liposomes and microbubbles,all of which are known in the art. For example, molecular imaging agentsincluding compositions of the present invention may be useful indiagnosing and/or evaluating a disease characterized by pathologicalangiogenesis as described herein.

Variant polypeptides of the present invention useful in the methods ofthe present invention include the following polypeptides: a polypeptidecomprising SEQ ID NO:25 wherein the amino acid of at least one ofpositions 149, 150, and 185 of SEQ ID NO:25 is an acidic amino acid, apolypeptide comprising SEQ ID NO:26 wherein the amino acid of at leastone of positions 123, 124, and 159 of SEQ ID NO:26 is an acidic aminoacid, and a polypeptide comprising SEQ ID NO:27 wherein the amino acidof at least one of positions 13, 14, and 49 of SEQ ID NO:27 is an acidicamino acid.

Variant polypeptides of the present invention useful in the methods ofthe present invention also include: a polypeptide comprising SEQ IDNO:25 wherein the amino acid at positions 149, 150, and 185 of SEQ IDNO:25 is an acidic amino acid, a polypeptide comprising SEQ ID NO:26wherein the amino acid at positions 123, 124, and 159 of SEQ ID NO:26 isan acidic amino acid, and a polypeptide comprising SEQ ID NO:27 whereinthe amino acid at positions 13, 14, and 49 of SEQ ID NO:27 is an acidicamino acid.

Variant polypeptides of the present invention useful in the methods ofthe present invention also include: a polypeptide comprising SEQ IDNO:6, a polypeptide comprising SEQ ID NO:7, a polypeptide comprising SEQID NO:8, a polypeptide comprising SEQ ID NO:10, a polypeptide comprisingSEQ ID NO:11, a polypeptide comprising SEQ ID NO:12, a polypeptidecomprising SEQ ID NO:14, a polypeptide comprising SEQ ID NO:15, apolypeptide comprising SEQ ID NO:16, a polypeptide comprising SEQ IDNO:18, a polypeptide comprising SEQ ID NO:19, a polypeptide comprisingSEQ ID NO:20, a polypeptide comprising SEQ ID NO:22, a polypeptidecomprising SEQ ID NO:23, or a polypeptide comprising SEQ ID NO:24.

In addition to the alterations at amino acid positions as describedherein, the VEGF variants of the present invention may contain furtheramino acid alterations, including substitutions and/or insertions and/ordeletions in any other region of the VEGF molecule, including the N- andC-terminal regions. Preferably, these substitutions will be“conservative” substitutions and do not alter the structure or functionof the resultant polypeptides based on either the native VEGF 165polypeptides of the present invention or the variant VEGF 165polypeptides of the present invention. The VEGF variants of the presentinvention show at least about 75%, at least about 80%, at least about85%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%, amino acid sequence identity with apolypeptide of the present invention. Such polypeptides include SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, or SEQ IDNO:24. Such polypeptides also include VEGF189 variant polypeptides ofthe present invention and VEGF isoform variant polypeptides which showat least about 75%, at least about 80%, at least about 85%, at leastabout 85%, at least about 86%, at least about 87%, at least about 88%,at least about 89%, at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, amino acid sequence identity with a polypeptide comprisingSEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62,SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68 and/or SEQ ID NO:69, and/or variants of each as describedherein.

An example of VEGF variants of the present invention which may containfurther amino acid alterations, including substitutions and/orinsertions and/or deletions in any other region of the VEGF molecule,including the N- and C-terminal region can be seen in FIG. 1A. FIG. 1A,shows orthologs of human VEGF165 which correspond to SEQ ID NO: 39through SEQ ID NO: 46. SEQ ID NO: 39 through SEQ ID NO: 46 are thenative amino acid sequence of orthologs of human VEGF165. Theseorthologs of human VEGF165 also contain the critical basic amino acid,arginine (Arg/R) residues which may be substituted with an acidic aminoacid, for example glutamic acid (Glu/E) to obtain the variants of thepresent invention. In particular, FIG. 1A shows orthologs of humanVEGF165 to F. catus (cat), where substitutions of an acidic amino acidin SEQ ID NO:39 could occur in at least one of R147, R148 and R183 ofSEQ ID NO: 39 which correspond to R121, R122, and R157 when consideringonly the part of SEQ ID NO:39 which reflects the amino acid sequence ofthe mature VEGF molecule; C. familiaris (dog), where substitutions of anacidic amino acid in SEQ ID NO:40 could occur in at least one of R148,R149 and R184 of SEQ ID NO: 40 which correspond to R122, R123, and R158when considering only the part of SEQ ID NO:40 which reflects the aminoacid sequence of the mature VEGF molecule; B. Taurus (bovine), wheresubstitutions of an acidic amino acid in SEQ ID NO:41 could occur in atleast one of R148, R149 and R184 of SEQ ID NO: 41 which correspond toR122, R123, and R158 when considering only the part of SEQ ID NO:41which reflects the amino acid sequence of the mature VEGF molecule; E.caballus (horse), R. norvegicus (rat), where substitutions of an acidicamino acid in SEQ ID NO:42 or SEQ ID NO:43 could occur in at least oneof R148, R149 and R184 of SEQ ID NO: 42 or SEQ ID NO: 43 whichcorrespond to R122, R123, and R158 when considering only the part of ofSEQ ID NO:42 or SEQ ID NO:43 which reflects the amino acid sequence ofthe mature VEGF molecule; M. musculus (mouse), where substitutions of anacidic amino acid in SEQ ID NO:44 could occur in at least one of R148,R149 and R184 of SEQ ID NO: 44 which correspond to R122, R123, and R158when considering only the part of SEQ ID NO:44 which reflects the aminoacid sequence of the mature VEGF molecule; G. gallus (chicken), wheresubstitutions of an acidic amino acid in SEQ ID NO:45 could occur in atleast one of R150, R151 and R186 of SEQ ID NO: 45 which correspond toR124, R125, and R160 when considering only the part of SEQ ID NO:45which reflects the amino acid sequence of the mature VEGF molecule; andX. laevis (frog), where substitutions of an acidic amino acid in SEQ IDNO:46 could occur in at least one of K150, R153 and R188 of SEQ ID NO:46 which correspond to K124, R127, and R162 when considering only thepart of SEQ ID NO:46 which reflects the amino acid sequence of themature VEGF molecule. Examples of 3S variants of these VEGF orthologswould be R121E/R122E/R157E (F. Catus), R122E/R123E/R159E (C. familiaris,B. Taurus, E. caballus, R. Norvegicus, M. Musculus, R124E/R125E/R160E(G. gallus) and K124E/R127E/R162E (X. laevis). These orthologs have highsequence identity with human VEGF variants of the present invention andshould be considered to be an example of VEGF variants of the presentinvention.

Variant polypeptides of the present invention include polypeptides whichshow at least about 75%, at least about 80%, at least about 85%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%, amino acid sequence identity with a polypeptidecomprising SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQID NO:43, SEQ ID NO:44, SEQ ID NO:45, and/or SEQ ID NO:46, and/orvariants of each as described herein.

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

In one embodiment, the present invention includes a polynucleotide thatencodes a variant polypeptide of the present invention. In oneembodiment, the present invention includes a polynucleotide that encodesa polypeptide comprising SEQ ID NO:25 wherein the amino acid of at leastone of positions 149, 150, and 185 of SEQ ID NO:25 is an acidic aminoacid. The present invention also includes a polynucleotide that encodesa polypeptide comprising SEQ ID NO:26 wherein the amino acid of at leastone of positions 123, 124, and 159 of SEQ ID NO:26 is an acidic aminoacid. The present invention also includes a polynucleotide that encodesa polypeptide comprising SEQ ID NO:27 wherein the amino acid of at leastone of positions 13, 14, and 49 of SEQ ID NO:27 is an acidic amino acid.

In other embodiments of the present invention includes a polynucleotidethat encodes a polypeptide comprising SEQ ID NO:6, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:7, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:8, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:10, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:11, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:12, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:14, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:15, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:16, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:18, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:19, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:20, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:22, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:23, or a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:24.

In another embodiment, the polynucleotide includes a polynucleotidecomprising SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, or SEQID NO:21. In another embodiment, the polynucleotide includes apolynucleotide comprising SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQID NO:31, SEQ ID NO:32, or SEQ ID NO:33.

The polynucleotides that encode VEGF variants of the present inventionmay contain further nucleotide alterations, including substitutionsand/or insertions and/or deletions in any other region of the VEGFmolecule, including the N- and C-terminal coding regions. Preferably,these substitutions will be “conservative” substitutions and do notalter the amino acid residues of the resultant polypeptides. In someembodiments, an amino acid residue may be altered, but the change is achange to another amino acid that is similar to the one replaced and thestructure and/or function of the resultant polypeptides will remain,based on either the native VEGF 165 polypeptides of the presentinvention or the variant VEGF 165 polypeptides of the present invention.The polynucleotides of the present invention which encode VEGF variantsof the present invention show at least about 75%, at least about 80%, atleast about 85%, at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99%, sequence identity with apolynucleotide which can be any of the following: a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:6, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:7, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:8, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:10, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:11, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:12, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:14, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:15, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:16, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:18, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:19, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:20, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:22, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:23, or a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:14; in another embodiment thepolynucleotide includes a polynucleotide that includes SEQ ID NO:1, SEQID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, or SEQ ID NO:21; inanother embodiment, the polynucleotide includes a polynucleotidecomprising SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQID NO:32, or SEQ ID NO:33.

In another embodiment, the polynucleotides of the present inventionwhich encode VEGF189 variants of the present invention show at leastabout 75%, at least about 80%, at least about 85%, at least about 85%,at least about 86%, at least about 87%, at least about 88%, at leastabout 89%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity with a polynucleotide a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:34, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:35, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:36, a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:37, or a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:38.

In another embodiment, the polynucleotides of the present inventionwhich encode other VEGF variants of the present invention show at leastabout 75%, at least about 80%, at least about 85%, at least about 85%,at least about 86%, at least about 87%, at least about 88%, at leastabout 89%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity with a polynucleotide a polynucleotide thatencodes a polypeptide comprising SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68 and/or SEQ ID NO:69, and/or variants of each asdescribed herein.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide

Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I.Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.);“Handbook of Experimental Immunology” (D. M. Weir & C. C. Blackwell,eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller & M. P.Calos, eds., 1987); “Current Protocols in Molecular Biology” (F. M.Ausubel et al., eds., 1987); “PCR: The Polymerase Chain Reaction”,(Mullis et al., eds., 1994); and “Current Protocols in Immunology” (J.E. Coligan et al., eds., 1991).

VEGF variants with more than one amino acid substitution may begenerated in one of several ways. If the amino acids are located closetogether in the polypeptide chain, they may be mutated simultaneously,using one oligonucleotide that codes for all of the desired amino acidsubstitutions. If, however, the amino acids are located some distancefrom one another (e.g. separated by more than ten amino acids), it ismore difficult to generate a single oligonucleotide that encodes all ofthe desired changes. Instead, one of two alternative methods may beemployed. In the first method, a separate oligonucleotide is generatedfor each amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions. The alternative methodinvolves two or more rounds of mutagenesis to produce the desiredmutant.

Amino acid sequence variants of the polypeptides of this invention(referred to in herein as the target polypeptide) are prepared byintroducing appropriate nucleotide changes into the DNA encoding thetarget polypeptide, or by in vitro synthesis of the desired targetpolypeptide. Any combination of deletion, insertion, and substitutioncan be made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics. The amino acid changesalso may alter post-translational processes of the target polypeptide,such as changing the number or position of glycosylation sites, alteringany membrane anchoring characteristics, and/or altering theintra-cellular location of the target polypeptide by inserting,deleting, or otherwise affecting any leader sequence of the nativetarget polypeptide.

There are two principal variables in the construction of amino acidsequence variants: the location of the mutation site and the nature ofthe mutation. In general, the location and nature of the mutation chosenwill depend upon the target polypeptide characteristic to be modified.

Amino acid sequence deletions of VEGF polypeptides of the presentinvention are generally not preferred, as maintaining the generallyconfiguration of a polypeptide is believed to be desirable for itsactivity. Any deletions will be selected so as to preserve thestructure.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the target polypeptide sequence) may range generallyfrom about 1 to 10 residues, more preferably 1 to 5, most preferably 1to 3. Examples of terminal insertions include the target polypeptidewith an N-terminal methionyl residue, an artifact of the directexpression of target polypeptide in bacterial recombinant cell culture,and fusion of a heterologous N-terminal signal sequence to theN-terminus of the target polypeptide molecule to facilitate thesecretion of the mature target polypeptide from recombinant host cells.Such signal sequences generally will be obtained from, and thushomologous to, the intended host cell species. Suitable sequencesinclude STH or Ipp for E. coli, alpha factor for yeast, and viralsignals such as herpes gD for mammalian cells. Other insertionalvariants of the target polypeptide include the fusion to the N- orC-terminus of the target polypeptide of immunogenic polypeptides, e.g.,bacterial polypeptides such as beta-lactamase or an enzyme encoded bythe E. coli trp locus, or yeast protein, and C-terminal fusions withproteins having a long half-life such as immunoglobulin constant regions(or other immunoglobulin regions), albumin, or ferritin.

DNA encoding amino acid sequence variants of the VEGF variants of thepresent invention is prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the target polypeptide.These techniques may utilize polypeptide nucleic acid (DNA or RNA), ornucleic acid complementary to the target polypeptide nucleic acid.

Oligonucleotide-mediated mutagenesis is one method for preparingsubstitution, deletion, and insertion variants of target polypeptideDNA. This technique is well known in the art as described by Adelman etal., DNA, 2: 183 (1983). Briefly, the target polypeptide DNA is alteredby hybridizing an oligonucleotide encoding the desired mutation to a DNAtemplate, where the template is the single-stranded form of a plasmid orbacteriophage containing the unaltered or native DNA sequence of thetarget polypeptide. After hybridization, a DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will code for theselected alteration in the target polypeptide DNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.Natl. Acad. Sci. USA, 75: 5765 (1978)). Single-stranded DNA template mayalso be generated by denaturing double-stranded plasmid (or other) DNAusing standard techniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase1, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of the target polypeptide, and the other strand (the originaltemplate) encodes the native, unaltered sequence of the targetpolypeptide. This heteroduplex molecule is then transformed into asuitable host cell, usually a prokaryote such as E. coli JM101. Afterthe cells are grown, they are plated onto agarose plates and screenedusing the oligonucleotide primer radiolabeled with 32-phosphate toidentify the bacterial colonies that contain the mutated DNA. Themutated region is then removed and placed in an appropriate vector forprotein production, generally an expression vector of the type typicallyemployed for transformation of an appropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(aS) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101, asdescribed above.

DNA encoding target polypeptide variants with more than one amino acidto be substituted may be generated in one of several ways. If the aminoacids are located close together in the polypeptide chain, they may bemutated simultaneously using one oligonucleotide that codes for all ofthe desired amino acid substitutions. If, however, the amino acids arelocated some distance from each other (separated by more than about tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant. The first round is as described for thesingle mutants: wild-type DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitutions) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on.

PCR mutagenesis is also suitable for making amino acid variants oftarget polypeptide. While the following discussion refers to DNA, it isunderstood that the technique also finds application with RNA. The PCRtechnique generally refers to the following procedure (see Erlich,supra, the chapter by R. Higuchi, p. 61-70): When small amounts oftemplate DNA are used as starting material in a PCR, primers that differslightly in sequence from the corresponding region in a template DNA canbe used to generate relatively large quantities of a specific DNAfragment that differs from the template sequence only at the positionswhere the primers differ from the template. For introduction of amutation into a plasmid DNA, one of the primers is designed to overlapthe position of the mutation and to contain the mutation; the sequenceof the other primer must be identical to a stretch of sequence of theopposite strand of the plasmid, but this sequence can be locatedanywhere along the plasmid DNA. It is preferred, however, that thesequence of the second primer is located within 200 nucleotides fromthat of the first, such that in the end the entire amplified region ofDNA bounded by the primers can be easily sequenced. PCR amplificationusing a primer pair like the one just described results in a populationof DNA fragments that differ at the position of the mutation specifiedby the primer, and possibly at other positions, as template copying issomewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer, or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more)-partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide tri-phosphates andis included in the GENEAMP kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayed with 35 μl mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Taq) DNA polymerase (5 units/μl, purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) programmed as follows: 2 minat 55° C., then 30 sec. at 72° C., then 19 cycles of the following: 30sec. at 94° C., 30 sec. at 55° C., and 30 sec. at 72° C. At the end ofthe program, the reaction vial is removed from the thermal cycler andthe aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50:vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to the appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. (Gene, 34: 315 (1985)). Thestarting material is the plasmid (or other vector) comprising the targetpolypeptide DNA to be mutated. The codon(s) in the target polypeptideDNA to be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the target polypeptide DNA. After therestriction sites have been introduced into the plasmid, the plasmid iscut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated target polypeptide DNA sequence.

In general, the signal sequence may be a component of the vector, or itmay be a part of the target polypeptide DNA that is inserted into thevector.

The polypeptides of this invention may be expressed not only directly,but also as a fusion with a heterologous polypeptide, preferably asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe target polypeptide DNA that is inserted into the vector. Includedwithin the scope of this invention are target polypeptides with anynative signal sequence deleted and replaced with a heterologous signalsequence. The heterologous signal sequence selected should be one thatis recognized and processed (i.e. cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe native target polypeptide signal sequence, the signal sequence issubstituted by a prokaryotic signal sequence selected, for example, fromthe group of the alkaline phosphatase, penicillinase, Ipp, orheat-stable enterotoxin II leaders. For yeast secretion, the nativetarget polypeptide signal sequence may be substituted by the yeastinvertase, alpha factor, or acid phosphatase leaders. In mammalian cellexpression the native signal sequence is satisfactory, although othermammalian signal sequences may be suitable.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are “shuttle” vectors, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

The present invention also relates to vectors comprising thepolynucleotide molecules of the invention, as well as host cellstransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be joined to a vector, which generally includes aselectable marker and an origin of replication, for propagation in ahost. Host cells are genetically engineered to express the polypeptidesof the present invention. The vectors include DNA encoding any of thepolypeptides described above or below, operably linked to suitabletranscriptional or translational regulatory sequences, such as thosederived from a mammalian, microbial, viral, or insect gene. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, mRNA ribosomal binding sites, and appropriate sequences whichcontrol transcription and translation. Nucleotide sequences are operablylinked when the regulatory sequence functionally relates to the DNAencoding the target protein. Thus, a promoter nucleotide sequences isoperably linked to polynucleotide of the present invention if thepromoter nucleotide sequence directs the transcription of thepolynucleotide of the present invention.

Selection of suitable vectors to be used for the cloning ofpolynucleotide molecules of the present invention will depend on thehost cell in which the vector will be transformed, and, whereapplicable, the host cell from which the target polypeptide is to beexpressed. Examples of vectors useful in the methods of the presentinvention include, but are not limited to, plasmids, bacteriophages,cosmids, retroviruses, and artificial chromosomes. Suitable host cellsfor expression of the polypeptides of the invention include prokaryotes,yeast, and higher eukaryotic cells.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of the target polypeptide DNA. However, the recovery ofgenomic DNA encoding the target polypeptide is more complex than that ofan exogenously replicated vector because restriction enzyme digestion isrequired to excise the target polypeptide DNA.

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, ortetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g. the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet., 1: 327(1982)), mycophenolic acid (Mulligan et al., Science, 209: 1422 (1980))or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 (1985)). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thetarget polypeptide nucleic acid, such as dihydrofolate reductase (DHFR)or thymidine kinase. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes the target polypeptide. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of the targetpolypeptide are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216 (1980). The transformed cells are then exposed toincreased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingthe target polypeptide. This amplification technique can be used withany otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1,notwithstanding the presence of endogenous DHFR if, for example, amutant DHFR gene that is highly resistant to Mtx is employed (EP117,060). Alternatively, host cells (particularly wild-type hosts thatcontain endogenous DHFR) transformed or co-transformed with DNAsequences encoding the target polypeptide, wild-type DHFR protein, andanother selectable marker such as aminoglycoside 3′ phosphotransferase(APH) can be selected by cell growth in medium containing a selectionagent for the selectable marker such as an aminoglycosidic antibiotic,e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979);Kingsman et al., Gene, 7: 141 (1979); or Tschemper et al., Gene, 10: 157(1980)). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)). The presence ofthe trp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the targetpolypeptide nucleic acid. Promoters are untranslated sequences locatedupstream (5′) to the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofa particular nucleic acid sequence, such as that encoding the targetpolypeptide, to which they are operably linked. Such promoters typicallyfall into two classes, inducible and constitutive. Inducible promotersare promoters that initiate increased levels of transcription from DNAunder their control in response to some change in culture conditions,e.g. the presence or absence of a nutrient or a change in temperature.At this time a large number of promoters recognized by a variety ofpotential host cells are well known. These promoters are operably linkedto DNA encoding the target polypeptide by removing the promoter from thesource DNA by restriction enzyme digestion and inserting the isolatedpromoter sequence into the vector. Both the native target polypeptidepromoter sequence and many heterologous promoters may be used to directamplification and/or expression of the target polypeptide DNA. However,heterologous promoters are preferred, as they generally permit greatertranscription and higher yields of expressed target polypeptide ascompared to the native target polypeptide promoter. Promoters suitablefor use with prokaryotic hosts include the β-lactamase and lactosepromoter systems (Chang et al., Nature, 275: 615 (1978); and Goeddel etal., Nature, 281: 544 (1979)), alkaline phosphatase, a tryptophan (trp)promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980) and EP36,776) and hybrid promoters such as the tac promoter (deBoer et al.,Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)). However, other knownbacterial promoters are suitable. Their nucleotide sequences have beenpublished, thereby enabling a skilled worker operably to ligate them toDNA encoding the target polypeptide (Siebenlist et al., Cell, 20: 269(1980)) using linkers or adaptors to supply any required restrictionsites. Promoters for use in bacterial systems also generally willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the target polypeptide.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg., 7: 149 (1968); and Holland, Biochemistry, 17: 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other yeast promoters, which are inducible promoters havingthe additional advantage of transcription controlled by growthconditions, are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, metallothionein, glyceraldehyde-3-phosphatedehydrogenase, and enzymes responsible for maltose and galactoseutilization. Suitable vectors and promoters for use in yeast expressionare further described in Hitzeman et al., EP 73,657A. Yeast enhancersalso are advantageously used with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

Target polypeptide transcription from vectors in mammalian host cells iscontrolled by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g. the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with thetarget polypeptide sequence, provided such promoters are compatible withthe host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209: 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78: 7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene, 18: 355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature, 295: 503-508 (1982) on expressing cDNA encoding immuneinterferon in monkey cells; Reyes et al., Nature, 297: 598-601 (1982) onexpression of human .beta.-interferon cDNA in mouse cells under thecontrol of a thymidine kinase promoter from herpes simplex virus,Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170 (1982) onexpression of the human interferon .beta.1 gene in cultured mouse andrabbit cells, and Gorman et al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

Transcription of DNA encoding the target polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10-300 bp, that act on a promoter to increase itstranscription. Enhancers are relatively orientation and positionindependent having been found 5′ (Laimins et al., Proc. Natl. Acad. Sci.USA, 78: 993 (1981)) and 3′ (Lusky et al., Mol. Cell Bio., 3: 1108(1983)) to the transcription unit, within an intron (Banerji et al.,Cell, 33: 729 (1983)) as well as within the coding sequence itself(Osborne et al., Mol. Cell Bio., 4:1293 (1984)). Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,.alpha.-fetoprotein and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature, 297: 17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the target polypeptide DNA, but is preferablylocated at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the target polypeptide. The 3′ untranslatedregions also include transcription termination sites.

Construction of suitable vectors containing one or more of the abovelisted components the desired coding and control sequences employsstandard ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9: 309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65: 499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding the target polypeptide. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Transientexpression systems, comprising a suitable expression vector and a hostcell, allow for the convenient positive identification of polypeptidesencoded by cloned DNAs, as well as for the rapid screening of suchpolypeptides for desired biological or physiological properties. Thus,transient expression systems are particularly useful in the inventionfor purposes of identifying analogs and variants of the targetpolypeptide that have target polypeptide-like activity.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the target polypeptide in recombinant vertebrate cellculture are described in Gething et al., Nature, 293: 620-625 (1981);Mantei et al., Nature, 281: 40-46 (1979); Levinson et al.; EP 117,060;and EP 117,058. A particularly useful plasmid for mammalian cell cultureexpression of the target polypeptide is pRK5 (EP pub. no. 307,247) orpSVI6B.

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes include eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, E. coli, Bacilli such as B. subtilis,Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, orSerratia marcescans. One preferred E. coli cloning host is E. coli 294(ATCC 31,446), although other strains such as E. coli B, E. coli 1776(ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. Theseexamples are illustrative rather than limiting. Preferably the host cellshould secrete minimal amounts of proteolytic enzymes. Alternatively, invitro methods of cloning, e.g. PCR or other nucleic acid polymerasereactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for target polypeptide-encodingvectors. Saccharomyces cerevisiae, or common baker's yeast, is the mostcommonly used among lower eukaryotic host microorganisms. However, anumber of other genera, species, and strains are commonly available anduseful herein, such as Schizosaccharomyces pombe (Beach and Nurse,Nature, 290: 140 (1981); EP 139,383 published May 2, 1985),Kluyveromyces hosts (U.S. Pat. No. 4,943,529) such as, e.g., K. lactis(Louvencourt et al., J. Bacteriol., 737 (1983)), K. fragilis, K.bulgaricus, K. thermotolerans, and K. marxianus, yarrowia (EP 402,226),Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,28: 265-278 (1988)), Candida, Trichoderma reesia (EP 244,234),Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)), and filamentous fungi such as, e.g, Neurospora,Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112: 284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474(1984)) and A. niger (Kelly and Hynes, EMBO J. 4: 475-479 (1985)).

Suitable host cells for the expression of target polypeptide are derivedfrom multicellular organisms. Such host cells are capable of complexprocessing and glycosylation activities. In principle, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori hostcells have been identified. See, e.g., Luckow et al., Bio/Technology 6:47-55 (1988); Miller et al., in Genetic Engineering, Setlow, J. K. etal., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda etal., Nature, 315: 592-594 (1985). A variety of such viral strains arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can beutilized as hosts. Typically, plant cells are transfected by incubationwith certain strains of the bacterium Agrobacterium tumefaciens, whichhas been previously manipulated to contain the target polypeptide DNA.During incubation of the plant cell culture with A. tumefaciens, the DNAencoding target polypeptide is transferred to the plant cell host suchthat it is transfected, and will, under appropriate conditions, expressthe target polypeptide DNA. In addition, regulatory and signal sequencescompatible with plant cells are available, such as the nopaline synthasepromoter and polyadenylation signal sequences. Depicker et al., J. Mol.Appl. Gen., 1: 561 (1982). In addition, DNA segments isolated from theupstream region of the T-DNA 780 gene are capable of activating orincreasing transcription levels of plant-expressible genes inrecombinant DNA-containing plant tissue. See EP 321,196 published Jun.21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years (Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36: 59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomacell line (Hep G2). Preferred host cells are human embryonic kidney 293and Chinese hamster ovary cells.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO.sub.4 and electroporation. Successful transfection isgenerally recognized when any indication of the operation of this vectoroccurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 publishedJun. 29, 1989. For mammalian cells without such cell walls, the calciumphosphate precipitation method described in sections 16.30-16.37 ofSambrook et al, supra, is preferred. General aspects of mammalian cellhost system transformations have been described by Axel in U.S. Pat. No.4,399,216 issued Aug. 16, 1983. Transformations into yeast are typicallycarried out according to the method of Van Solingen et al., J. Bact.,130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829(1979). However, other methods for introducing DNA into cells such as bynuclear injection, electroporation, or protoplast fusion may also beused.

Prokaryotic cells used to produce the target polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the target polypeptide of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enz., 58: 44(1979), Barnes and Sato, Anal. Biochem., 102: 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195;U.S. Pat. No. Re. 30,985, may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

It is further envisioned that the target polypeptides of this inventionmay be produced by homologous recombination, or with recombinantproduction methods utilizing control elements introduced into cellsalready containing DNA encoding the target polypeptide currently in usein the field. For example, a powerful promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredtarget polypeptide. The control element does not encode the targetpolypeptide of this invention, but the DNA is present in the host cellgenome. One next screens for cells making the target polypeptide of thisinvention, or increased or decreased levels of expression, as desired.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201-5205 (1980)), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, particularly .sup.32 P. However, othertechniques may also be employed, such as using biotin-modifiednucleotides for introduction into a polynucleotide. The biotin thenserves as the site for binding to avidin or antibodies, which may belabeled with a wide variety of labels, such as radionuclides,fluorescers, enzymes, or the like. Alternatively, antibodies may beemployed that can recognize specific duplexes, including DNA duplexes,RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native target polypeptide or against a synthetic peptide basedon the DNA sequences provided herein as described further in Section 4below.

The target polypeptide preferably is recovered from the culture mediumas a secreted polypeptide, although it also may be recovered from hostcell lysates when directly expressed without a secretory signal.

When the target polypeptide is expressed in a recombinant cell otherthan one of human origin, the target polypeptide is completely free ofproteins or polypeptides of human origin. However, it is necessary topurify the target polypeptide from recombinant cell proteins orpolypeptides to obtain preparations that are substantially homogeneousas to the target polypeptide. As a first step, the culture medium orlysate is centrifuged to remove particulate cell debris. The membraneand soluble protein fractions are then separated. The target polypeptidemay then be purified from the soluble protein fraction and from themembrane fraction of the culture lysate, depending on whether the targetpolypeptide is membrane bound. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; and protein A Sepharosecolumns to remove contaminants such as IgG.

Target polypeptide variants in which residues have been deleted,inserted or substituted are recovered in the same fashion, takingaccount of any substantial changes in properties occasioned by thevariation. For example, preparation of a target polypeptide fusion withanother protein or polypeptide, e.g. a bacterial or viral antigen,facilitates purification; an immunoaffinity column containing antibodyto the antigen (or containing antigen, where the target polypeptide isan antibody) can be used to adsorb the fusion. Immunoaffinity columnssuch as a rabbit polyclonal anti-target polypeptide column can beemployed to absorb the target polypeptide variant by binding it to atleast one remaining immune epitope. A protease inhibitor such as phenylmethyl sulfonyl fluoride (PMSF) also may be useful to inhibitproteolytic degradation during purification, and antibiotics may beincluded to prevent the growth of adventitious contaminants. One skilledin the art will appreciate that purification methods suitable for nativetarget polypeptide may require modification to account for changes inthe character of the target polypeptide or its variants upon expressionin recombinant cell culture.

The VEGF variants of the present invention can be administered fortherapeutic treatments to a patient suffering from a tumor orangiogenesis associated pathologic condition in an amount sufficient toprevent, inhibit, or reduce the progression of the tumor or pathologiccondition. Progression includes, e.g, the growth, invasiveness,metastases and/or recurrence of the tumor or pathologic condition. Anamount adequate to accomplish this is defined as a therapeuticallyeffective dose. Amounts effective for this use will depend upon theseverity of the disease and the general state of the patient's ownimmune system. Dosing schedules will also vary with the disease stateand status of the patient, and will typically range from a single bolusdosage or continuous infusion to multiple administrations per day (e.g.,every 4-6 hours), or as indicated by the treating physician and thepatient's condition. It should be noted, however, that the presentinvention is not limited to any particular dose.

In an embodiment of the invention, VEGF variants of the presentinvention can be administered in combination with one or more otherantineoplastic agents. Any suitable antineoplastic agent can be used,such as a chemotherapeutic agent or radiation. When the antineoplasticagent is radiation, the source of the radiation can be either external(external beam radiation therapy—EBRT) or internal (brachytherapy—BT) tothe patient being treated. VEGF variant polypeptides of the presentinvention can be administered together with a antineoplastic agent whichincludes a chemotherapeutic agent. A “chemotherapeutic agent” is achemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclosphosphamide, alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nirnustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin, dynemicin, including dynemicinA; an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,pteropterin, trimeterxate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgenssuch as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate;CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoic acid; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

In one embodiment, the combination therapy can include combinations withVEGF variants of the present invention and, such as, for example, IFL(5-fluorouracil, leucovorin and irinotecan), FOLFOX (5-fluorouracil,leukovorin and oxaliplatin); XELOX (capecitabine and oxaliplatin),paclitaxel, docetaxel, (capecitabine, taxane, and antracycline),(carboplatin and paclitaxel), (cisplatin and gemcitabine), erlotinib,interferon-2α, (carboplatin and paclitaxel), sunitinib, sorafenib,pazopanib, (vandefanib and paclitaxel), cetuximab, (oxaliplatin- oririnotecan-based chemotherapy and panitumumab), capecitabine,(capecitine or 5-fluorouracil and cisplatin), gemcitabine, (gemcitabineand erlotinib), (docetaxel and prednisone), prednisone, pemetrexed,(5-fluorouracil, leukovorin and irinotecan), (leukovorin and5-fluorouracil), lomustine, bevacizumab, aflibercept, sunitinib,sorafenib, PTK787, semaxanib, axitinib, vandetanib, cediranib.

The dose of antineoplastic agent administered depends on numerousfactors, including, for example, the type of agent, the type andseverity tumor being treated and the route of administration of theagent. It should be emphasized, however, that the present invention isnot limited to any particular dose.

Further, VEGF variants of the present invention may be administered withantibodies that neutralize other receptors involved in tumor growth orangiogenesis. One such receptor is EGFR. In an embodiment of the presentinvention, a VEGF variant of the present invention is used incombination with an EGFR antagonist. An EGFR antagonist can be anantibody that binds to EGFR or a ligand of EGFR and inhibits binding ofEGFR to its ligand. Examples of EGFR antagonists that bind EGFR include,without limitation, biological molecules, such as antibodies (andfunctional equivalents thereof) specific for EGFR, and small molecules,such as synthetic kinase inhibitors that act directly on the cytoplasmicdomain of EGFR. Other examples of growth factor receptors involved intumorigenesis are the receptors for platelet-derived growth factor(PDGFR), insulin-like growth factor (IGFR), nerve growth factor (NGFR),and fibroblast growth factor (FGFR).

In an additional alternative embodiment, the VEGF variants of thepresent invention can be administered in combination with one or moresuitable adjuvants, such as, for example, cytokines (IL-10 and IL-13,for example) or other immune stimulators. See, e.g., Larrivee et al.,supra. It should be appreciated, however, that administration of only ananti-KDR antibody is sufficient to prevent, inhibit, or reduce theprogression of the tumor in a therapeutically effective manner.

In a combination therapy, a VEGF variant of the present invention isadministered before, during, or after commencing therapy with anotheragent, as well as any combination thereof, i.e., before and during,before and after, during and after, or before, during and aftercommencing the antineoplastic agent therapy. For example, a VEGF variantof the present invention may be administered between 1 and 30 days,preferably 3 and 20 days, more preferably between 5 and 12 days beforecommencing radiation therapy.

It is noted that VEGF variants of the present invention can beadministered as a conjugate, which binds specifically to the receptorand delivers a toxic, lethal payload following ligand-toxininternalization.

The present invention also includes kits for inhibiting tumor growthand/or angiogenesis comprising a therapeutically effective amount of oneor more of a VEGF variant of the present invention. The kits can furthercontain any suitable antagonist of, for example, another growth factorreceptor involved in tumorigenesis or angiogenesis (e.g., EGFR, PDGFR,IGFR, NGFR, FGFR, etc, as described above). Alternatively, or inaddition, the kits of the present invention can further comprise anantineoplastic agent. Examples of suitable antineoplastic agents in thecontext of the present invention have been described herein. The kits ofthe present invention can further comprise an adjuvant, examples havealso been described above.

It is understood that VEGF variants of the present invention, where usedin a mammal for the purpose of prophylaxis or treatment, will beadministered in the form of a composition additionally comprising apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include, for example, one or more of water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. Pharmaceutically acceptable carriers mayfurther comprise minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the binding proteins. The compositions of theinjection may, as is well known in the art, be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the mammal.

Pharmaceutical compositions of the present invention can comprise apolynucleotide encoding a VEGF variant herein, or, alternatively,pharmaceutical compositions can comprise the VEGF variant itself.

Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, inhalation, or by injection. For example,pharmacological agents or compositions injected into the blood streamshould be soluble. Other factors are known in the art, and includeconsiderations such as toxicity and forms that prevent the agent orcomposition from exerting its effect.

Compositions comprising a VEGF variant or a polynucleotide encoding aVEGF variant can also be formulated as pharmaceutically acceptable salts(e.g., acid addition salts) and/or complexes thereof. Pharmaceuticallyacceptable salts are non-toxic at the concentration at which they areadministered. Pharmaceutically acceptable salts include acid additionsalts such as those containing sulfate, hydrochloride, phosphate,sulfonate, sulfamate, sulfate, acetate, citrate, lactate, tartrate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,cyclohexylsulfonyl, cyclohexylsulfamate and quinate. Pharmaceuticallyacceptable salts can be obtained from acids such as hydrochloric acid,sulfuric acid, phosphoric acid, sulfonic acid, sulfamic acid, aceticacid, citric acid, lactic acid, tartaric acid, malonic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, cyclohexylsulfonic acid, cyclohexylsulfamicacid, and quinic acid. Such salts may be prepared by, for example,reacting the free acid or base forms of the product with one or moreequivalents of the appropriate base or acid in a solvent or medium inwhich the salt is insoluble, or in a solvent such as water which is thenremoved in vacuo or by freeze-drying or by exchanging the ions of anexisting salt for another ion on a suitable ion exchange resin.

Carriers or excipients can also be used to facilitate administration ofthe compound. Examples of carriers and excipients include calciumcarbonate, calcium phosphate, various sugars such as lactose, glucose,or sucrose, or types of starch, cellulose derivatives, gelatin,vegetable oils, polyethylene glycols and physiologically compatiblesolvents. The compositions or pharmaceutical composition can beadministered by different routes including, but not limited to,intravenous, intra-arterial, intraperitoneal, intrapericardial,intracoronary, subcutaneous, and intramuscular, oral, topical, ortransmucosal.

The desired isotonicity of the compositions can be accomplished usingsodium chloride or other pharmaceutically acceptable agents such asdextrose, boric acid, sodium tartrate, propylene glycol, polyols (suchas mannitol and sorbitol), or other inorganic or organic solutes.

Pharmaceutical compositions comprising a VEGF variant or apolynucleotide encoding a VEGF variant can be formulated for a varietyof modes of administration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co.,Easton, Pa. 1990. See, also, Wang and Hanson “Parenteral Formulations ofProteins and Peptides: Stability and Stabilizers”, Journal of ParenteralScience and Technology, Technical Report No. 10, Supp. 42-2S (1988). Asuitable administration format can best be determined by a medicalpractitioner for each patient individually.

For systemic administration, injection is preferred, e.g.,intramuscular, intravenous, intra-arterial, intracoronary,intrapericardial, intraperitoneal, subcutaneous, intrathecal, orintracerebrovascular. For injection, the compounds of the invention areformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. Alternatively, thecompounds of the invention are formulated in one or more excipients(e.g., propylene glycol) that are generally accepted as safe as definedby USP standards. They can, for example, be suspended in an inert oil,suitably a vegetable oil such as sesame, peanut, olive oil, or otheracceptable carrier. Preferably, they are suspended in an aqueouscarrier, for example, in an isotonic buffer solution at pH of about 5.6to 7.4. These compositions can be sterilized by conventionalsterilization techniques, or can be sterile filtered. The compositionscan contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions, such as pH buffering agents.Useful buffers include for example, sodium acetate/acetic acid buffers.A form of repository or “depot” slow release preparation can be used sothat therapeutically effective amounts of the preparation are deliveredinto the bloodstream over many hours or days following transdermalinjection or delivery. In addition, the compounds can be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

Alternatively, the compounds can be administered orally. For oraladministration, the compounds are formulated into conventional oraldosage forms such as capsules, tablets and tonics.

Systemic administration can also be by transmucosal or transdermal. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, bile salts and fusidic acid derivatives. Inaddition, detergents can be used to facilitate permeation. Transmucosaladministration can be, for example, through nasal sprays or usingsuppositories.

For administration by inhalation, usually inhalable dry powercompositions or aerosol compositions are used, where the size of theparticles or droplets is selected to ensure deposition of the activeingredient in the desired part of the respiratory tract, e.g. throat,upper respiratory tract or lungs. Inhalable compositions and devices fortheir administration are well known in the art. For example, devices forthe delivery of aerosol medications for inspiration are known. One suchdevice is a metered dose inhaler that delivers the same dosage ofmedication to the patient upon each actuation of the device. Metereddose inhalers typically include a canister containing a reservoir ofmedication and propellant under pressure and a fixed volume metered dosechamber. The canister is inserted into a receptacle in a body or basehaving a mouthpiece or nosepiece for delivering medication to thepatient. The patient uses the device by manually pressing the canisterinto the body to close a filling valve and capture a metered dose ofmedication inside the chamber and to open a release valve which releasesthe captured, fixed volume of medication in the dose chamber to theatmosphere as an aerosol mist. Simultaneously, the patient inhalesthrough the mouthpiece to entrain the mist into the airway. The patientthen releases the canister so that the release valve closes and thefilling valve opens to refill the dose chamber for the nextadministration of medication. See, for example, U.S. Pat. No. 4,896,832and a product available from 3M Healthcare known as Aerosol SheathedActuator and Cap.

For topical administration, the compounds of the invention areformulated into ointments, salves, gels, or creams, as is generallyknown in the art. If desired, solutions of the above compositions can bethickened with a thickening agent such as methyl cellulose. They can beprepared in emulsified form, either water in oil or oil in water. Any ofa wide variety of pharmaceutically acceptable emulsifying agents can beemployed including, for example, acacia powder, a non-ionic surfactant(such as a Tween), or an ionic surfactant (such as alkali polyetheralcohol sulfates or sulfonates, e.g., a Triton).

Compositions useful in the invention are prepared by mixing theingredients following generally accepted procedures. For example, theselected components can be mixed simply in a blender or other standarddevice to produce a concentrated mixture which can then be adjusted tothe final concentration and viscosity by the addition of water orthickening agent and possibly a buffer to control pH or an additionalsolute to control tonicity.

The amounts of various compounds for use in the methods of the inventionto be administered can be determined by standard procedures. Generally,a therapeutically effective amount is between about 100 mg/kg and 10⁻¹²mg/kg depending on the age and size of the patient, and the disease ordisorder associated with the patient.

The present invention also provides delivery vehicles suitable fordelivery of a polynucleotide encoding a VEGF variant into cells (whetherin vivo, ex vivo, or in vitro). Generally, a polynucleotide encoding aVEGF variant will be operably linked to a promoter and a heterologouspolynucleotide. A polynucleotide encoding a VEGF variant can becontained within a cloning or expression vector, using methods wellknown in the art, or within a viral vector. These vectors (especiallyexpression vectors) can in turn be manipulated to assume any of a numberof forms, which may, for example, facilitate delivery to and/or entryinto a target cell. Delivery of the polynucleotide constructs of theinvention to eukaryotic cells, particularly to mammalian cells, moreparticularly to distal tubule cells of the kidney, can be accomplishedby any suitable art-known method. Delivery can be accomplished in vivo,ex vivo, or in vitro.

The invention provides methods and compositions for transferring suchexpression constructs into cells, especially in vivo for performing themethods of the present invention. It is also an object of the inventionto provide compositions for the treatment (including prevention) of theconditions listed above by providing for the prevention or repair of theunderlying vascular injury and/or the associated damage to non-vasculartissues. Delivery vehicles suitable for incorporation of apolynucleotide encoding a VEGF variant of the present invention forintroduction into a host cell include non-viral vehicles and viralvectors. Verma and Somia (1997) Nature 389:239-242.

A wide variety of non-viral vehicles for delivery of a polynucleotideencoding a VEGF variant are known in the art and are encompassed in thepresent invention. A polynucleotide encoding a VEGF variant can bedelivered to a cell as naked DNA (U.S. Pat. No. 5,692,622; WO 97/40163).Alternatively, a polynucleotide encoding a VEGF variant can be deliveredto a cell associated in a variety of ways with a variety of substances(forms of delivery) including, but not limited to cationic lipids;biocompatible polymers, including natural polymers and syntheticpolymers; lipoproteins; polypeptides; polysaccharides;lipopolysaccharides; artificial viral envelopes; metal particles; andbacteria. A delivery vehicle can be a microparticle. Mixtures orconjugates of these various substances can also be used as deliveryvehicles. A polynucleotide encoding a VEGF variant can be associatednon-covalently or covalently with these various forms of delivery.Liposomes can be targeted to a particular cell type, e.g., to aglomerular epithelial cell.

Viral vectors include, but are not limited to, DNA viral vectors such asthose based on adenoviruses, herpes simplex virus, poxviruses such asvaccinia virus, and parvoviruses, including adeno-associated virus; andRNA viral vectors, including, but not limited to, the retroviralvectors. Retroviral vectors include murine leukemia virus, andlentiviruses such as human immunodeficiency virus. Naldini et al. (1996)Science 272:263-267.

Non-viral delivery vehicles comprising a polynucleotide encoding a VEGFvariant can be introduced into host cells and/or target cells by anymethod known in the art, such as transfection by the calcium phosphatecoprecipitation technique; electroporation; electropermeabilization;liposome-mediated transfection; ballistic transfection; biolisticprocesses including microparticle bombardment, jet injection, and needleand syringe injection; or by microinjection. Numerous methods oftransfection are known to the skilled worker in the field.

Viral delivery vehicles can be introduced into cells by infection.Alternatively, viral vehicles can be incorporated into any of thenon-viral delivery vehicles described above for delivery into cells. Forexample, viral vectors can be mixed with cationic lipids (Hodgson andSolaiman (1996) Nature Biotechnol. 14:339-342); or lamellar liposomes(Wilson et al. (1977) Proc. Natl. Acad. Sci. USA 74:3471; and Faller etal. (1984) J. Virol. 49:269). For in vivo delivery, the deliveryvehicle(s) can be introduced into an individual by any of a number ofmethods, each of which is familiar in the art.

The present invention also includes a method for creating a polypeptidecapable of inhibiting angiogenesis by: (1) providing a native VEGFcomprising a C-terminal heparin binding domain and (2) modifying saidnative VEGF to form a variant VEGF of the invention.

Further details of the present invention will be apparent from thefollowing non-limiting Examples. All references cited throughout thespecification, including the Examples, are hereby expressly incorporatedby reference.

EXAMPLES Example 1

293/KDR cells were from SibTech and maintained as described (Backer etal., 2005). Heparin and biotin heparin was from Sigma. Purified HStetramer and HS oligomer were from Neoparin.

The following Gateway Entry (Invitrogen) clones (all 3.8 kB) weregenerated: Hs.VEGF165 Wild Type sequence (SEQ ID NO:28); Hs.VEGF165R149E (SEQ ID NO:33); Hs.VEGF165 R150E (SEQ ID NO:32); Hs.VEGF165 R185E(SEQ ID NO:31); Hs.VEGF165 R149E/R150E (SEQ ID NO:30); Hs.VEGF165R149E/R150E/R185E (SEQ ID NO:29); all plasmids were grown in E. coliwith spectrinomycin (100 ug/ml) for plasmid selection.

Plasmids for mammalian expression of VEGF165 wild type and mutant forms(all 6.1 kB) under the control of a CMV/T7 promoter were generated fromGateway Entry clones (Invitrogen). All possess ampicillin resistanceused for plasmid propagation (100 ug/ml), neomycin (G418) resistance formammalian selection (500-800 ug/ml, depending on target cell type), thenative VEGF165 signal peptide sequence and G418 resistance. Thefollowing clones of VEGF were generated for mammalian expression:Hs.VEGF165 wild type sequence; Hs.VEGF165 Wild Type sequence (SEQ IDNO:28); Hs.VEGF165 R149E (SEQ ID NO:33); Hs.VEGF165 R150E (SEQ IDNO:32); Hs.VEGF165 R185E (SEQ ID NO:31); Hs.VEGF165 R149E/R150E (SEQ IDNO:30); Hs.VEGF165 R149E/R150E/R185E (SEQ ID NO:29).

For proliferation assays, 293/KDR derived cell lines (5×10⁴ cellsperwell) were seeded in 6-well culture plates in quadruplicate. Cell numberper well was measured after 2, 3, 4, and 5 days of growth by removingcells with trypsin, collecting via centrifugation and counting suspendedcells in triplicate in a Cellometer Automatic Cell Counter (NexcelomBioscience, Lawrence, Mass.). Purified recombinant VEGF-A protein usedas an external standard quantitation as VEGF mass per mass totalextracted cell protein. KDR autophosphorylation in cell lysates wasmeasured similarly; cultured cells were serum-deprived for 48 h, wherenoted cells were stimulated for 20 min with WT or 3S VEGF-A alone or incombination at the indicated concentrations, then extracted with icecold buffer containing non-ionic detergent, protease and phosphataseinhibitors. Cleared extracts were applied to assay plates and processingper the manufacturer's instructions.

KDR and VEGF content in cell lysates, tissue extracts and conditionedmedia was determined using two-site electrochemiluminescent immunoassaysdeveloped by Meso Scale Discovery (Gaithersburg, Md.) for use with theMeso Scale Discovery (MSD) SectorImager 2400 plate reader.

KDR autophosphorylation in cell lysates or tumor tissue extracts wasmeasured similarly but included parallel detection with anti-receptorantibodies and specific anti-phospho-receptor antibodies or 4G10(Millipore). Cell lysates were prepared as described previously (Athaudaet al., 2006). Tumor tissue non-ionic detergent extracts were preparedusing the same buffer but accompanied by physical disruption in aMini-BeadBeater-8 (Glen Mills Inc.) and clearing by centrifugation priorto immunoassay.

Ligand binding assays were also developed on the electrochemiluminescentplatform. KDR-Ig ectodomain saturation binding to VEGF165 WT or 3Sproteins that had been captured using a non-neutralizing monoclonal VEGFantibody; bound KDR was detected using tagged anti-KDR. All measurementswere made on triplicate samples. GraphPad Prism software version 5.0 wasused for statistical analyses and determination of K_(D) and IC₅₀values.

Example 2

To determine whether 3S VEGF 165 (SEQ ID NO:7) retained mitogenicactivity relative to the WT VEGF 165 sequence, we stably transfectedHEK293 cells that had been engineered to express 2.5×10⁶ VEGFR2 per cell(293/KDR; 22) with expression plasmids encoding vector alone, WT or 3SVEGF 165. Ectopic VEGF protein production by transfectants was measuredusing a two-site immunoassay with a detection limit of 37.5 attomoles/25ul (1.5 pM) VEGF (FIG. 3).

Among marker selected mass cultures, WT transfectants produced ˜1.0ng/ml/24 h VEGF165 protein in conditioned media, 3S transfectantsproduced ˜2.5 ng/ml/24 h, and VEGF protein was undetectable in the emptyvector control media (FIG. 4A, white bars). The VEGF content in lowvolume detergent extracts from the same cells was proportionally higher,as expected (FIG. 4A, gray bars). Molecular mass and antibodyrecognition of VEGF165 3S protein in conditioned media wereindistinguishable from WT (FIG. 4B). Saturation binding of VEGF165 3S toKDR in vitro (FIG. 4C, squares; KD ˜19 pM) was also equivalent toVEGF165 WT binding (FIG. 4C, circles; KD ˜23 pM) and consistent withpublished steady-state binding affinity values (Ferrara, 2004).

KDR tyrosyl phosphorylation in each cell line was measured after 24 h ofserum deprivation (FIG. 4D). The basal KDR phosphorylation level inempty vector transfectants (FIG. 4D, white bar) was indistinguishablefrom that of the parental cell line (not shown) or the 3S transfectants(FIG. 4D, dark gray bar). In contrast, WT transfectant basal KDRphosphorylation level was 4-fold higher than control or 3S (FIG. 4D,light gray bar; p<0.001). The KDR autophosphorylation level of thevector transfectant after 20 min exposure to exogenously added VEGF165protein (2.5 nM) is shown for reference (FIG. 4D, black bar). Consistentwith the levels of KDR activation among the transfectants, significantdifferences in cell proliferation rate in culture were observed from day3 onward: WT transfectants (FIG. 4E, squares) grew significantly fasterthan the vector control (FIG. 4E, circles) or 3S transfectants (FIG. 4E,triangles; p<0.001 between WT and control or WT and 3S for days 3-5).These results suggested that the WT transfectants had acquired autocrineVEGF signaling, but not the 3S transfectants, even though 3S proteinproduction was more than twice that of WT. In soft agar colony formationassays, empty vector transfectants and S3 transfectants grew modestly,if at all (FIG. 4F, left and middle panels), whereas VEGF165 WTtransfectants grew robustly (FIG. 4F, right panel). All of these resultsindicated loss of signaling by VEGF165 3S, despite normal KDR binding.

Competitive antagonism of VEGF165 WT signaling by VEGF165 3S protein wasassessed in intact cells and in vivo (FIG. 5). Concentrated conditionedmedia harvested from the 3S transfectants was added to 293/KDR cells inthe presence of purified VEGF165 WT protein and phospho-KDR levels weremeasured (FIGS. 5A and B). Since the conditioned media could containother inhibitors of KDR activation, VEGF165 3S was selectively removedfrom the media by immunodepletion with anti-VEGF antibody. Amock-immunodepletion was performed in parallel using a non-specificantibody and the VEGF165 3S content of the anti-VEGF andmock-immunodepleted media was measured (FIG. 5A). VEGF 3S levels innon-immunodepleted and mock immunodepleted media were nearly identical(1.93 and 1.89 ng/mg total cell protein, respectively; FIG. 5A, whitebars), while immunodepletion with anti-VEGF removed 95% of the 3Sprotein (0.096 ng/mg total cell protein; FIG. 5A, gray bar). VEGFprotein was not detected in media from empty vector transfectants beforeor after immunodepletion (FIG. 5A, right). KDR autophosphorylationstimulated by purified VEGF165 protein (10 ng/ml) in serum-deprived293/KDR cells (FIG. 5B, 100%) was inhibited modestly byVEGF-immunodepleted media (FIG. 5B, circles), but mock-immunodepletedmedia showed significant, dose-dependent inhibition, with >80%inhibition by media containing 2.5-fold molar excess VEGF 3S protein(FIG. 5B, triangles). In soft agar colony formation assays, the robustanchorage independent growth of 293/KDR cells stably transfected withVEGF165 WT (FIG. 5C, upper left panel) was inhibited in a dose-dependentmanner by pazopanib, a VEGFR-selective antagonist (FIG. 5C, middle andright upper panels), providing additional evidence that colony formationwas driven by autocrine VEGF/KDR signaling. VEGF165 3S protein added at0.06, 0.15 and 0.6 nM also resulted in significant, dose-dependentinhibition (FIG. 5C, lower panels).

To test whether VEGF165 3S protein could antagonize KDR-driventumorigenicity in mice, animals were implanted subcutaneously withVEGF165 WT transfected 293/KDR cells (3×10⁶ per animal), with the samenumber of VEGF165 3S transfected cells, or with a suspension containing1.5×10⁶ cells of each line. Additional control groups received the emptyvector 293/KDR cells (3×10⁶ per animal) or the empty vector cellscombined with VEGF WT transfectants at 1.5×10⁶ cells each. The lattergroup indicated the growth rate of tumors arising from 1.5×10⁶ VEGF WTtransfectants in the presence of “neutral” cells providing the sameinitial mass; a growth rate below this threshold in the group implantedwith VEGF WT+VEGF 3S transfectants could be attributed to inhibition ofVEGF WT-driven tumor growth by VEGF165 3S. Indeed, VEGF WT transfected293/KDR cells formed tumors fastest (FIG. 5D, squares), whereas VEGF 3Stransfectants did not form tumors prior to study termination (FIG. 5D,inverted triangles), and animals implanted with the WT+3S cell mix (FIG.5D, diamonds) formed tumors at a significantly lower rate than thecontrol WT+empty vector group (FIG. 5D, triangles) throughout the study(p<0.05). The ability of the parental cell line (FIG. 5D, circles) toform tumors in mice, in the absence of robust soft agar colonyformation, suggests that endogenous murine VEGF drove tumorigenesis in aparacrine manner, and the failure of the 3S transfectants (FIG. 5D,triangles) to form tumors suggests antagonism of this pathway. Theanimal studies also indicate that autocrine VEGF/KDR-driventumorigenesis by the VEGF165 WT transfectants (FIG. 5D, squares),potentially enhanced by murine VEGF, was competitively antagonized bysecreted VEGF165 3S protein in animals receiving the WT+3S transfectantcell mix (FIG. 5D, diamonds).

Example 3

For proliferation assays, 293/KDR derived cell lines were seeded in6-well culture plates in quadruplicate. Cell number per well wasmeasured at regular intervals removal of adherent cells and counting ina Cellometer Automatic Cell Counter (Nexcelom Bioscience). Assays forcolony formation in soft agar were performed as described (Castagnino etal., 2000).

SoftAgar Colony Formation Assays for colony formation in soft agar(representative of anchorage independent growth) were performed andquantitated as described previously. Briefly, U87 MG cells and U87 MGcells transfected with a plasmid encoding NK1 60/62/73 were suspended in0.5% Seaplaque agarose (1×105 cells) in growth medium in duplicate 60 mmdishes. Cell colonies were visualized after staining withp-iodotetrazolium violet by brightfield microscopy using an Olympusinverted microscope. Digital images were acquired and recorded using aCCD video camera and IP Lab software. Consistent with prior results,293/KDR cells stably transfected with an expression plasmid for WTVEGF165 displayed robust anchorage independent growth. Treatment withpazopanib, a VEGFR-selective competitive antagonist of ATP binding everyother day at 0.3 and 3.0 nM over two weeks resulted in substantial anddose-dependent inhibition of colony formation, providing additionalevidence that it was primarily driven by autocrine VEGF/KDR signaling.Treatment with 3S VEGF protein at 0.06, 0.15, and 0.6 nM also resultedin substantial and dose-dependent inhibition, consistent with theobserved inhibition of KDR kinase activation.

Example 4

All experiments involving animals were performed in accordance with NIHGuidelines for Care and Use of Laboratory Animals using approvedprotocols. 293/KDR cells stably transfected with VEGF WT or 3Sexpression plasmids, or empty vector, were injected subcutaneously intoSCID/Beige mice (Taconic, Inc.; 3×10⁶ cells per animal total asindicated in the text, 5 mice/group) and tumor volumes were measured atregular intervals as described previously (Giubellino et al., 2007).Statistical analysis and curve fitting for all animal studies wereperformed using GraphPad Prism software version 5.0.

To test whether 3S VEGF could antagonize KDR-driven tumorigenicity inmice, groups of animals (n=5) were implanted subcutaneously with WT VEFGtransfected 293/KDR cells (3×10⁶ per animal), with the same number of 3SVEGF transfected cells, or with a suspension containing half the numberof cells of each type. Additional control groups received the emptyvector 293/KDR cells (3×10⁶ per animal) or the empty vector cellscombined with WT VEGF transfectants at 1.5×10⁶ cells each. The lattergroup indicted the growth rate of tumors arising from 1.5×10⁶ WT VEGFtransfectants in the presence of “neutral” cells providing the sameinitial mass; a growth rate below this threshold in the group implantedwith WT VEGF+3S VEGF transfectants could be attributed to inhibition by3S VEGF. Indeed, WT VEGF transfected 293/KDR cells formed tumorsfastest, 3S VEGF transfectants tumors grew most slowly, and animalsimplanted with the WT VEGF+ 3S VEGF transfectant mixture formed tumorsat a significantly lower rate that the control WT VEGF+empty vectorgroup throughout the study (paired two-tailed t test P=0.0108, t=4.497,df=4). These results strongly suggest that autocrine VEGF/KDR-driventumorigenesis by the WT VEFG transfectants was competitively antagonizedby secreted 3S VEGF protein. Together with our prior results with 3SHGF/NK1, the current work reinforces the theory that receptor occupancycombined with disruption of ternary HS binding may constitute aneffective general strategy for antagonizing signaling by HS bindinggrowth factors.

Example 5

The possibility that VEGF 3S had lost biological activity due to loss ofits dimeric tertiary structure was investigated by SDS-PAGE andimmunoblot analysis. We found that, like VEGF165 WT, VEGF 3S dimers andmonomers of expected mass were detected under non-reducing and reducingconditions, respectively (FIG. 7). FIG. 7 shows VEGF165 3S (VEGF 3S;left) and VEGF165 WT (VEGF WT; middle) proteins in 24 h conditionedmedia prepared from 293/KDR transfectants, and purified recombinantVEGF165 protein (VEGF 4 ng; right), after SDS-PAGE under non-reducing(NR) and reducing (R) conditions and immunoblotting with anti-VEGF.Migration of molecular mass standards (kDa) is indicated by arrows. Inaddition to evidence already present in the application, these resultsstrengthen the likelihood that changes engineered to the VEGF HS bindingdomain did not affect its tertiary structure.

As anticipated, found that heparin promoted clustering of VEGF165 WT,but not of VEGF165 3S (FIG. 8). FIG. 8 shows heparin-Sepharose mediatedclustering of VEGF165 WT and VEGF165 3S analyzed by SDS-PAGE andimmunodetection with anti-VEGF antibody. VEGF165 WT (left) or VEGF165 3S(right) conditioned media contained VEGF dimers (44 kDa) in bothsupernatant (super) and Heparin-Sepharose (HS beads) samples only in thepresence of BS3. An 80 kDa complex consistent with tetrameric VEGF (andhigher mass species of indeterminant copy number) was observed only inVEGF165 WT conditioned media in the presence of heparin (4th lane fromleft) and not in VEGF165 3S conditioned media (4th lane from the right).These findings further support the mechanism of action of VEGF 3S asantagonism of HS binding to the VEGF/VEGFR complex leading toinstability of ligand/receptor/HS complexes and consequent disruption ofdownstream signaling.

Analysis of the spectrum of VEGF 3S antagonism showed that VEGF165 3Sinhibited placenta growth factor (P1GF)-induced Akt activation(phospho-Akt/total Akt) in EA.hy 926 cells, which express VEGFR1 and R2,with potency similar to pazopanib (FIG. 9A). In contrast, VEGF 3S didnot block Akt activation induced by VEGF-D in SCC-25 cells, whichexpress VEGFR1, R2 and R3 (FIG. 9B, C). Thus the pattern of VEGF 3Sinhibition followed the pattern of VEGF-A binding to VEGFR1 and R2, butnot homodimers of VEGFR3, as anticipated. FIG. 9(A) shows dose dependentinhibition of VEGF-or P1GF-induced Akt activation (phospho Akt/totalAkt) in EA.hy 926 cells. VEGF 3s blocked Akt activation by VEGF-A (darkcircles) or P1GF (squares). VEGF 2S blocked Akt activation by VEGF-A(dark circles) or P1GF (squares) with potency similar to pazopanib(triangles and inverted triangles, respectively). FIG. 9(B) showsdose-dependent Akt activation (phosphor-Akt/total Akt) was measured byelectrochemilumnescent two-site immunoassays in detergent extractsprepared from intact, serum deprived SCC-25 squamous cell carcinomacells stimulated with VEGF165 (10-1000 pM) or VEGFD (100-10,000 pM) for15 minutes at 37 degrees C. SCC-25 cells express all three VEGFRs. FIG.9(C) shows VEGF165 3S antagonism of Akt activation by VEGF-A (at 100 pM)but not VEGF-D (at 1000 pM). These results suggest that VEGF165 3Santagonism follows the pattern of VEGF-A binding as anticipated: VEGF-Abinding to VEGFR1 and R2 should be antagonized, whereas VEGF-D bindingto VEGFR3 homodimers should not be affected.

VEGF165 3S antagonism was found to be independent of NRP1-VEGF-Aprotein-protein interaction. NRP1 binds to the VEGF-A HS binding domainprimarily at R165 and secondarily at K147, E152 and E155, i.e., to asurface opposite that of HS. Crosslinking studies further showed thatVEGF165 WT and VEGF165 3S bound similarly to NRP1 in vitro (FIG. 10A).Functionally, VEGF165 3S antagonized VEGF signaling similarly in 293/KDRderived cells, which lack NRP1 (FIG. 10B, lanes 4-6), and in EA.hy 926cells which are NRP1 positive (FIG. 10B, lane 2).

In FIG. 10(A) Covalent affinity crosslinking analysis of NRP1 binding toVEGF165 WT and VEGF165 3S. Purified recombinant NRP1-Fc (Sino Biologics)was added as indicated (−NRP1 or +NRP1) to 24 h conditioned media fromvector transfected 293/KDR cells(left lanes), 293/KDR/VEGF 3Stransfectants (middle lanes), or 293/KDR/VEGF WT transfectants (rightlanes) in the absence (−) or presence (+) of the crosslinking reagentBS3 (Thermo Fisher Pierce). Samples were resolved by SDS-PAGE underreducing conditions, transferred to PVDF and immunodetected with anti-Fcantibody (top panel; Thermo Fisher Pierce) or anti-VEGF (lower panels;R&D Systems). A 160 kDa band, consistent with a 1:1 complex of NRP1-Fcand VEGF dimer, was detected by both antibodies specifically in thepresence of NRP1-Fc in media from 293/KDR/VEGF 3S transfectants and293/KDR/VEGF WT transfectants but not the vector control. Another bandof approximately 250 kDa was observed with the same pattern; thestoichiometry of this complex is indeterminant. Monomeric (22 kDa) ispresent in all lanes where VEGF media was added, and serves as a loadingcontrol. Dimeric VEGF (44 kDa) is captured from both 293/KDR/VEGF 3Smedia and 293/KDR/VEGF WT media only in the presence of BS3 in thereducing SDS-PAGE conditions used.

FIG. 10(B) The relative abundance of NRPI protein (upper panel) in PC3prostate adenocarcinoma cells (lane 1), EA.hy 926 cells (lane 2), HEK293cells (lane 3), 293/KDR vector transfected cells (lane 4),293/KDR/VEGF165 WT cells (lane 5) and 293/KDR/VEGF165 3S cells (lane 6),determined by SDS-PAGE and immunoblotting with anti-NRPI (CellSignaling). The bottom panel shows a GAPDH immunoblot of the samesamples as a loading control.

Experimental Procedures

Reagents and Cell Culture: Purified VEGF proteins and antibodies forVEGF165 and CD44 were from R&D Systems, ABR Affinity Bioreagent andSanta Cruz Biotech. Pazopanib was from Tocris. Heparin and biotinheparin were from Sigma; HS tetramer and oligomer were from Neoparin.The cell lines 184B5, U87 MG, SK-LMS-1, B16, PC3M, EA.hy 926 and SCC-25were cultured as described (Athauda et al., 2006; Giubellino et al.,2007; Lalla et al., 2003). 293/KDR cells from SibTech and maintained asdescribed (Backer et al., 2005). Transfection reagents were from AmaxaBiosystems.

Plasmids for VEGF165: Plasmids for mammalian expression of VEGF165 WT(R123, R124 and R159) and VEGF165 3S (R123E/R124E/R159E) under thecontrol of a CMV promoter were generated from Gateway Entry clones(Invitrogen) and possess the native VEGF165 signal peptide sequence andG418 resistance.

Immunoassays: KDR and VEGF content in cell lysates, tissue extracts andconditioned media was determined using 2-site electrochemiluminescentimmunoassays developed for use with the Meso Scale Discovery (MSD)SectorImager 2400 plate reader (Gaithersburg, Md.). KDR activation incell lysates or tumor tissue extracts included parallel detection withanti-receptor antibodies and specific anti-phospho-receptor antibodiesor 4G10 (Millipore). Cell lysates were prepared similarly for analysisof activated (phospho-) Akt, and total Akt using immunoassays from MSD.All measurements were made on triplicate samples. GraphPad Prismsoftware version 5.0 was used for all statistical analyses: proteincontent values determined by 2-site immunoassay were interpolated fromstandard curves by nonlinear regression analysis (4 parameter logisticequation); saturation and competitive binding curves were fit afterdetermination of specific binding and assuming a one site binding model.

Covalent Affinity Crosslinking: Assays. Heparin-mediated clustering ofVEGF165 WT and VEGF165 3S was investigated using concentratedconditioned media from 293/KDR/VEGF165 WT or 293/KDR/VEGF165 3Stransfectants incubated at 40 C for 16 h in the presence of HeparinSepharose 6 Fast Flow (GE Healthcare). Heparin Sepharose beads were usedas both a source of heparin and to efficiently capture heparin-clusteredVEGF from the conditioned media after crosslinking. Samples were thenincubated with or without BS3 at room temperature for 1 h. Afterpelleting the beads by brief centrifugation, samples were obtained fromthe supernatants for SDS-PAGE and immunoblot analysis. The beads werethen washed by five centrifugation/resuspension cycles in PBS, and boundproteins were eluted with boiling SDS-PAGE sample buffer. Supernatantand bead-captured samples were then resolved by SDS-PAGE under reducingconditions, transferred to PVDF and immunodetected with anti-VEGFantibody.

1. A method for treating a disease characterized by pathologicalangiogenesis, comprising administering to a patient in need thereof apharmaceutically effective amount of a vascular endothelial cell growthfactor (VEGF) variant polypeptide comprising (1) a polypeptidecomprising a polypeptide selected from the group consisting of SEQ IDNO:25 wherein the amino acid of at least one of positions 149, 150, and185 of SEQ NO:25 is an acidic amino acid, SEQ ID NO:26 wherein the aminoacid of at least one of positions 123, 124, and 159 of SEQ ID NO:26 isan acidic amino acid, and a polypeptide comprising SEQ NO:27 wherein theamino acid of at least one of positions 13, 14, and 49 of SEQ NO:27 isan acidic amino acid; or (2) a polypeptide comprising a polypeptidehaving at least 95% identity to a polypeptide of (1) and having theability to antagonize VEGF mediated angiogenesis.
 2. The method of claim1 wherein the affinity of the variant polypeptide for both VEGFR-1(FLT-1) and VEGFR-2 (KDR/FLK-1) is substantially maintained incomparison to said native VEGF.
 3. (canceled)
 4. (canceled)
 5. Themethod of claim 1, wherein the acidic amino acid is E or D.
 6. Themethod of claim 1, wherein the variant polypeptide is selected from thegroup consisting of: (1) a polypeptide comprising a polypeptide selectedfrom the group consisting of SEQ ID NO:25 wherein the amino acid atpositions 149, 150, and 185 of SEQ ID NO:25 is an acidic amino acid, SEQID NO:26 wherein the amino acid at positions 123, 124, and 159 of SEQ IDNO:26 is an acidic amino acid, and SEQ ID NO:27 wherein the amino acidat positions 13, 14, and 49 of SEQ ID NO:27 is an acidic amino acid; and(2) a polypeptide having at least 95% identity to a polypeptide of (1)and having the ability to antagonize VEGF mediated angiogenesis.
 7. Themethod of claim 6, wherein the acidic amino acid is E or D.
 8. Themethod of claim 1, wherein the variant polypeptide is selected from thegroup consisting of: (1) a polypeptide comprising a polypeptide selectedfrom the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:23, and SEQ ID NO:24; and (2) a polypeptide having at least 95%identity to a polypeptide of (1) and having the ability to antagonizeVEGF mediated angiogenesis.
 9. The method of claim 1, wherein thevariant polypeptide comprises SEQ ID NO:7.
 10. (canceled)
 11. (canceled)12. The method of claim 1, wherein the variant polypeptide is selectedfrom the group consisting of: (1) a polypeptide comprising the matureform of a polypeptide selected from the group consisting of SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; and(2) a polypeptide having at least 95% identity to a polypeptide of (1)and having the ability to antagonize VEGF mediated angiogenesis. 13.(canceled)
 14. (canceled)
 15. The method of claim 1, wherein the diseasecharacterized by pathological angiogenesis is cancer, maculardegeneration, or diabetic retinopathy.
 16. The method of claim 15,wherein the cancer is a metastatic cancer.
 17. A method for creating apolypeptide capable of inhibiting angiogenesis comprising: (a) providinga native VEGF comprising a C-terminal heparin binding domain and (b)modifying said native VEGF to form a variant VEGF, wherein the variantVEGF is selected from the group consisting of: (1) a polypeptidecomprising a polypeptide selected from the group consisting of SEQ IDNO:25 wherein the amino acid of at least one of positions 149, 150, and185 of SEQ ID NO:25 is an acidic amino acid, SEQ ID NO:26 wherein theamino acid of at least one of positions 123, 124, and 159 of SEQ IDNO:26 is an acidic amino acid, and SEQ ID NO:27 wherein the amino acidof at least one of positions 13, 14, and 49 of SEQ ID NO:27 is an acidicamino acid; and (2) a polypeptide having at least 95% identity to apolypeptide of (1) and having the ability to antagonize VEGF mediatedangiogenesis. 18-27. (canceled)
 28. A vascular endothelial cell growthfactor (VEGF) variant polypeptide comprising (1) a polypeptidecomprising a polypeptide selected from the group consisting of SEQ IDNO:25 wherein the amino acid of at least one of positions 149, 150, and185 of SEQ ID NO:25 is an acidic amino acid, SEQ ID NO:26 wherein theamino acid of at least one of positions 123, 124, and 159 of SEQ IDNO:26 is an acidic amino acid, and SEQ ID NO:27 wherein the amino acidof at least one of positions 13, 14, and 49 of SEQ ID NO:27 is an acidicamino acid; or (2) a polypeptide comprising a polypeptide having atleast 95% identity to a polypeptide of (1) and having the ability toantagonize VEGF mediated angiogenesis.
 29. (canceled)
 30. The variantpolypeptide of claim 28, wherein the acidic amino acid is E or D. 31.The variant polypeptide of claim 28, wherein the variant polypeptide isselected from the group consisting of: (1) a polypeptide comprising apolypeptide selected from the group consisting of SEQ ID NO:25 whereinthe amino acid at positions 149, 150, and 185 of SEQ ID NO:25 is anacidic amino acid, SEQ ID NO:26 wherein the amino acid at positions 123,124, and 159 of SEQ ID NO:26 is an acidic amino acid, SEQ ID NO:27wherein the amino acid at positions 13, 14, and 49 of SEQ ID NO:27 is anacidic amino acid; and (2) a polypeptide having at least 95% identity toa polypeptide of (1) and having the ability to antagonize VEGF mediatedangiogenesis.
 32. The variant polypeptide of claim 31, wherein theacidic amino acid is E or D.
 33. The variant polypeptide of claim 28,wherein the variant polypeptide is selected from the group consistingof: (1) a polypeptide comprising a polypeptide selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24; and (2) a polypeptide having at least 95% identity to apolypeptide of (1) and having the ability to antagonize VEGF mediatedangiogenesis.
 34. The variant polypeptide of claim 28, wherein thevariant polypeptide comprises SEQ ID NO:7. 35-44. (canceled)